WO2024092015A1 - Inhibition de molécules d'adhésion pour thérapies par cellules souches - Google Patents

Inhibition de molécules d'adhésion pour thérapies par cellules souches Download PDF

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WO2024092015A1
WO2024092015A1 PCT/US2023/077747 US2023077747W WO2024092015A1 WO 2024092015 A1 WO2024092015 A1 WO 2024092015A1 US 2023077747 W US2023077747 W US 2023077747W WO 2024092015 A1 WO2024092015 A1 WO 2024092015A1
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cells
cell
psc
stem cells
hypoimmune
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Matthew Brown
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Wisconsin Alumni Research Foundation
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    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70503—Immunoglobulin superfamily
    • C07K14/70525—ICAM molecules, e.g. CD50, CD54, CD102
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/14—Blood; Artificial blood
    • A61K35/17—Lymphocytes; B-cells; T-cells; Natural killer cells; Interferon-activated or cytokine-activated lymphocytes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
    • A61L27/38—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70503—Immunoglobulin superfamily
    • C07K14/70539—MHC-molecules, e.g. HLA-molecules
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    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
    • C12N5/0657—Cardiomyocytes; Heart cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
    • A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
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    • C12N2502/00—Coculture with; Conditioned medium produced by
    • C12N2502/45—Artificially induced pluripotent stem cells
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    • C12N2506/00—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells
    • C12N2506/45—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells from artificially induced pluripotent stem cells
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
    • C12N9/22—Ribonucleases RNAses, DNAses

Definitions

  • pluripotent stem cell (PSC)-derived cell therapies are promising reparative treatments for a variety of cardiovascular diseases that kill over 655,000 Americans each year.
  • PSC-dcrivcd grafts have multiple uniquely attractive attributes, such as near-infinite scalability and a lack of passenger lymphocytes, and they may have diminished susceptibility to the acute and chronic allograft rejection that routinely devastates traditional organ transplantations.
  • PSCs are amenable to CRISPR/Cas9-based gene-editing.
  • hypoimmune “universal cells” e.g., knock-out [KO] of HLA class I and/or II
  • hypoimmune PSC grafts in patients, including whether such a dramatic intervention as total ablation of HLA I+II increases the risk of deleterious effects (e.g., malignancy, uncontrolled viral replication).
  • stem cell e.g., iPSC or embryonic stem cell
  • therapies that are functional and curative, and that reduce the risk of rejection by the recipient immune system. This will enable long-term graft function and improvement of patient lives.
  • an in vitro method of preparing a population of hypoimmune mammalian stem cells comprises providing a population of isolated mammalian stem cells, wherein the isolated mammalian stem cells express a cell adhesion molecule; and modifying the expression of the cell adhesion molecule in the population of isolated mammalian stem cells to decrease or knockout expression of the cell adhesion molecule and provide the population of hypoimmune mammalian stem cells.
  • a population of hypoimmune mammalian stem cells wherein expression of a cell adhesion molecule is decreased or knocked out by modification by gene editing of a gene for the cell adhesion molecule.
  • the hypoimmune mammalian stem cells can be differentiated to endothelial cells, cardiac cells, fibroblasts, pancreatic cells, neural cells, or islet cells.
  • a graft comprising the population of differentiated hypoimmune mammalian stem cells and a method of treating a mammalian subject by introducing the graft.
  • Figures 1A and B show an RNA sequencing analysis of wild-type and B2M KO Hl PSC-AEC cell culture models (CMs) of allorejection inflammation.
  • CMs cell culture models
  • FIGS. 2A and B show transplantation of gene-edited PSC-CVTs into immune- deficient mouse hosts.
  • (2B) CMs made from B2M KO Hl PSCs were formed into spheroids and transplanted heterotopically into the receptive kidney capsule transplant site of a NBGSW mouse. After 28 days in vivo, the animal was anesthetized and the graft was examined macroscopically, showing robust contraction and neovascularization with human vessels.
  • Figure 3 shows direct and indirect pathway of alloreactivity in first-generation hypoimmune PSC-AECs.
  • Hl WT and B2M-K0 PSCs were differentiated into AEC targets and co-cultured for 6 days with HLA-mismatched peripheral blood leukocytes (10:1 E:T ratio). Alloreactivity was assessed via proliferation (CFSE dye dilution) of CD4+ and CD8+ cells.
  • B2M-KO PSCs showed diminished but still present CD8+ proliferation, indicating indirect pathway alloreactivity.
  • Figures 4A and B show assessment of human T cell phenotype and PSC immunogenicity in the NeoThy humanized mouse.
  • (4A) Flow cytometric analysis of NeoThy and BLT peripheral blood for naive T cell marker CD45RA in CD3+CD4+ populations. BLT mice have a significantly higher (p>0.0001) percentage of naive cells, which is less representative of normal patient frequencies.
  • (4B) iPSC-derived CMs from HLA-B/HLA-DR mismatched donor transplanted under the NeoThy kidney capsule, IHC staining at d26 posttransplantation.
  • FIG. 5 shows flow cytometry phenotyping of PSC-CMs.
  • Figure 8 shows bioluminescent imaging (BLI) using Akaluc reporter.
  • PSC-AECs harboring a constitutively expressed Akaluc reporter were injected into the hind limb of an NBSGW mouse and monitored for retention.
  • Figure 9 shows in vitro Maturation of PSC-CMs. 98% cTNT+ PSC-CMs were cultured in vitro and harvested for RNA at two timepoints, early (14d, post-0 days extra culture) and late (14d+28 days extra culture. Differential gene expression is shown for those genes related to CM specification and maturation.
  • Figure 10 shows sequencing-based TCR rearrangement analysis. FACS sorted CD3+CD8+ T cells from two patients (Pl and P2) were analyzed for baseline ex vivo TCR beta chain clonal diversity with the Adaptive Biotechnologies ImmunoSEQTM kit.
  • Figure 11A shows the Crispr gene editing scheme (SEQ ID NO: 5 is the ICAM1 DNA sequence, SEQ ID NO: 6 is the ICAM1 KO ssODN, and SEQ ID NO: 7 is the gRNA).
  • FIG. 12 shows CD54 KO PSCs are capable of differentiation into highly-pure CMs.
  • CMs were assessed for cardiac troponin-T via intracellular flow cytometry at day 17 of differentiation.
  • WT (left) and KO (right) were similar in purity.
  • Figure 13 A shows tri-cellular PSC-CVTs in culture. Contracting spheroids made from fused PSC-CMs, PSCC-fibs, and ECs contract in culture (4x image).
  • FIG. 13B shows RNAseq data: including lack of redundancy of other ICAMs (e.g., ICAM2) making up for ICAM1 loss, indications that the KO has downregulated HLA, which may contribute to diminished immunogenicity/rejection response, and additional insights from the past month of data analysis in KO PSCs and CMs.
  • ICAM2 ICAM2
  • KEGG Analysis H9 WT CMs were compared to H9 CD54 KO CMs. Multiple KEGG pathways were enriched in WT vs KO cells, including extracellular matrix-receptor interaction, focal adhesion, AGE-RAGE signaling and PI3K-Akt signaling pathways. These pathways play critical roles in immune cell interaction, and indicate that in KO cells the innate immune cells may be directly impacted, which in turn could impact adaptive immune responses i.e., diminished innate and adaptive immune responses to KO cell grafts.
  • Figure 14A shows post-transplant photos of H9 CD54KO CMs. Graft is apparent, with clear evidence of neovascularization and engraftment. 14B)shows post-transplant photos of multi-cellular cardiovascular grafts. Grafts contain CMs, endothelial cells, smooth muscle cells, and cardiac fibroblasts derived from PSCs.
  • FIGS 15A and B show efficient generation and differentiation of ICAM-1 KO and isogenic WT PSCs.
  • ICAM-1 was ablated via homozygous CRISPR/Cas9 KO.
  • KO and PSCs were stimulated with IFNy and TNFa for 48 hours, showing intact HLA class I but absence of ICAM-1 in KO.
  • 15B Cells were differentiated into highly pure (>85%) (left) cardiac troponin T (cTNT)+ CMs and (right) CD31 + CD144 + CD184 + AECs. (Unstained cells used as comparison controls, subgating of additional AEC markers-data not shown).
  • Figure 16 illustrates an assay for assessing direct and indirect pathway of alloreactivity of PSC-AECs.
  • Hl WT and B2M-K0 PSCs were differentiated into AEC targets and co-cultured for 6 days with HLA-mismatched peripheral blood leukocytes (10: 1 E:T ratio). Alloreactivity was assessed via proliferation (CFSE dye dilution) of CD4 + and CD8 + cells.
  • B2M- KO PSCs showed diminished but still present CD8+ proliferation, indicating indirect pathway alloreactivity.
  • Figure 18 shows PBMC binding to WT vs ICAM-1 KO PSCs.
  • PSCs were incubated for 1 hour with fluorescently-labeled allogeneic PBMCs, washed, and imaged to determine # of bound immune cells. Counts determined by blinded acquisition of four noncontiguous brightfield images prior to fluorescence imaging (lOx). Analysis via ImageJ.
  • FIG 19 shows an allorejection assay using bioluminescent imaging (BLI).
  • ICAM-1 knock-out (KO) PSCs IxlO 6 ) harboring a constitutively expressed Akaluc reporter were injected with Matrigel® into the right hind limb of humanized NeoThy mice.
  • Isogenic wild-type (WT) were injected into the left leg.
  • BLI signal was monitored for 32 days at an early, mid, and late/terminal timepoint. Representative mouse is shown reflecting loss of WT graft, and retention of KO, seen in 3 of 4 mice.
  • Figure 20 shows RNAseq data: including lack of redundancy of other ICAMs (e.g., ICAM2) making up for ICAM1 loss, indications that the KO has downrcgulatcd HLA, which may contribute to diminished immunogenicity/rejection response, and additional insights from the past month of data analysis in KO PSCs and CMs.
  • EdgeR Differential Gene Expression Analysis H9 WT CMs were compared to H9 CD54 KO CMs. Multiple genes were differentially upregulated in WT vs KO CMs. The top 10 most significantly upregulated genes are shown.
  • FIG. 21 shows RNAseq data: including lack of redundancy of other ICAMs (e.g., ICAM2) making up for ICAM1 loss, indications that the KO has downregulated HLA, which may contribute to diminished immunogenicity/rejection response, and additional insights from the past month of data analysis in KO PSCs and CMs.
  • H9 WT CMs were compared to H9 CD54 KO CMs. Multiple immune-associated genes were differentially upregulated in WT vs KO CMs. The top 19 most significantly upregulated genes are shown.
  • FIG. 22 shows RNAseq data: including lack of redundancy of other ICAMs (e.g., ICAM2) making up for ICAM1 loss, indications that the KO has downregulated HLA, which may contribute to diminished immunogenicity/rejection response, and additional insights from the past month of data analysis in KO PSCs and CMs.
  • H9 WT CMs were compared to H9 CD54 KO CMs. Multiple immune-associated genes were differentially upregulated in WT vs KO CMs. Higher levels of these genes on WT (and lower levels on KO), such as HLA-A and IL11, may confer immune protection of the KO cells.
  • FIG. 23A and B shows a multiplex Luminex® assay performed on H9 WT CMs and CD54 KO CMs and Validated by RNAseq.
  • MCP1 CCL2
  • Luminex® assay CCL2
  • n 3 biological replicates with 2 technical replicates each.
  • Figure 24 illustrates diminished immunogenicity associated with CD54 Knockout.
  • FIG. 25 shows Generation of CD54 Knockout Pluripotent Stem Cell Lines.
  • CD54 was knocked-out via CRISPR/Cas9.
  • the diagram indicates the KO strategy.
  • SEQ ID NO: 8 is the WT exon
  • SEQ ID NO: 9 is the edited exon).
  • Figures 26A and B show pluripotency of CD54 KO PSCs.
  • 26A Cells are positive for SSEA-4 by flow cytometry.
  • 26B Cells are alkaline phosphatase positive. For both A and B, isogenic WT and KO are show left and right, respectively. These data demonstrate that we have successfully generated multiple lines and they maintain pluripotency i.eterrorism are bone fide PSCs.
  • Figure 27 shows LFA-1 and MAC-1 Staining on Immune Cells. LFA-1 and MAC-1 are both ligands for ICAM-1 (CD54) and are present at differing levels on many immune cells.
  • peripheral blood lymphocytes left
  • monocytes right
  • monocytic lymphoma line U937 right
  • FIG. 28 shows blocking ICAM-1 Inhibits Immune Cell Binding.
  • PSCderived endothelial cells were stimulated with TNFa and IFNg for 48h, then ICAM-1 was blocked via antibody incubation.
  • Ecs were then co-cultured briefly with U937 monocytic leukemia cells and peripheral blood mononuclear cells (PBMCs, a mixture of lymphocytes and monocytes). The immune cells were washed off, with all wells being treated equally. Images of fluorescently labeled immune cells co-cultured with Ecs were acquired. Image J software was used to quantify bound cells. Five regions of interest were imaged and quantified, data summarized in plot. These data demonstrates that blocking ICAM-1 inhibits binding of LFA-1 and/or MAC-1 and thus diminishes binding of multiple types of immune cells.
  • FIGS 29A and B shows static Immune Cell Adhesion Assay.
  • H9 WT and CD54KO PSCs were differentiated into endothelial cells (ECs), stimulated with TNFa and IFNg for 48h, and then co-cultured briefly with U937 monocytic leukemia cells. The U937 cells were washed off, with all wells being treated equally.
  • the disclosure provides for improving allogeneic cell tolerance, including methods of making cells that have reduced immunogenicity after grafting, e.g., transplant, and methods of using those cells.
  • adhesion proteins on cells that immune cells use for attaching/binding to the cells, which is one of the first steps in the immune cell rejecting cells that are not self.
  • knocking expression of one or more of those proteins down or out in stem cells such as pluripotent stem cells was found to inhibit or prevent immune cells from attaching to differentiated cells arising from the modified stem cells that can be used as therapeutics.
  • the resulting cells can be used to treat a wide variety of diseases including, but not limited to cardiovascular disease, diabetes, neurological disorders, liver diseases, and viral infection.
  • the cells may be PSC-beta islets (diabetes), PSC-hepatocytes (liver disease), PSC-neurons and neural subtypes (neurological diseases), PSC-endothelial cells (vascular disease), however, any cell type may benefit from knocking out expression that results in diminished immune system recognition.
  • reduced immunogenicity means cells are less likely to be engaged with and/or rejected by any immune cell(s). Reduced immunogenicity can be shown by reduced binding to immune cells. Reduced immunogenicity can also be shown as a diminished proliferative response from T cells upon encountering ICAM-1 KO cells such as via mixed lymphocyte reaction (MLR). Further, in vivo in the NeoTHy humanized mouse, the KO cells persist longer than the WT cells. This means that the immune system does not recognize and reject them as well as it does the WT cells.
  • MLR mixed lymphocyte reaction
  • an in vitro method of preparing a population of hypoimmune mammalian stem cells comprises providing a population of isolated mammalian stem cells, wherein the isolated mammalian stem cells express a cell adhesion molecule; and modifying the expression of the cell adhesion molecule in the population of isolated mammalian stem cells to decrease or knockout expression of the cell adhesion molecule and provide the population of hypoimmune mammalian cells.
  • the population of hypoimmune mammalian cells is less immunogenic than the corresponding population of isolated mammalian stem cells, wherein less immunogenic is as defined above.
  • the population of isolated mammalian stem cells can comprise pluripotent stem cells, or embryonic stem cells, and can be human or non-human stem cells.
  • the one or more mammalian stem cells are non-human stem cells, e.g., non-human primate, bovine, equine, canine, feline, caprine, swine, murine, or ovine stem cells.
  • the knockout cells may be used in xenotransplantation studies, e.g., in addition to the various current gene edits that are made in pigs, a CD54 knockout may improve transplants and/or make them less likely to be rejected by human immune cells.
  • isolated immune evading (hypoimmune) stem cells are provided, e.g., human pluripotent stem cells such as induced human pluripotent stem cells (iPSCs) or human embryonic stem cells, which have decreased or lack expression (“knock down” or “knock out”) of one or more cell adhesion molecules, e.g., as a result of gene editing of one or more alleles (producing heterozygotes or homozygotes) or other approaches to inhibit expression such as inhibitory RNAs (siRNA, shRNA, miRNA, antisense oligonucleotides, etc.).
  • iPSCs induced human pluripotent stem cells
  • knock out a cell adhesion molecules
  • Gene editing can include CRISPR intervention, e.g., using Casl3 or dCas9, and the like.
  • a CRISPR gene edit may be employed to insert a stop codon in a gene, e.g., in an open reading frame, and thus prevent expression of a functional gene product.
  • the population of hypoimmune mammalian stem cells may be differentiated into any cell type or a plurality of cell types, e.g., differentiated into cardiomyocytes or endothelial cells, where the target adhesion protein(s) remained knocked down or knocked out in the differentiated cells.
  • adhesion proteins include, but are not limited to, CD54 (ICAM-1), ICAM-2, ICAM-3, ICAM-4, ICAM-5, VCAM, MADCAM-1, P-selectin, E-selectin, L-selectin, integrins, focal adhesion molecules, extracellular matrix molecules, co- stimulatory molecules, and other molecules involved in the immune synapse and/or tethering, rolling, and extravasation.
  • IAM-1 CD54
  • ICAM-2 ICAM-2
  • ICAM-3 ICAM-3
  • ICAM-4 ICAM-5
  • VCAM VCAM
  • MADCAM-1 MADCAM-1
  • P-selectin E-selectin
  • L-selectin L-selectin
  • integrins integrins
  • focal adhesion molecules extracellular matrix molecules
  • co- stimulatory molecules and other molecules involved in the immune synapse and/or tethering, rolling, and extravasation.
  • the isolated cells e.g., isolated stem cells such as pluripotent stem cells or differentiated cells, e.g., T cells or other immune cells, such as hematopoietic stem or progenitor cells, cardiomyocytes, fibroblasts, endothelial cells, lack or have reduced expression of CD54 or other adhesion molecule(s), or lack or have reduced expression of a combination of adhesion molecules.
  • isolated stem cells such as pluripotent stem cells or differentiated cells, e.g., T cells or other immune cells, such as hematopoietic stem or progenitor cells, cardiomyocytes, fibroblasts, endothelial cells
  • CD54 or other adhesion molecule(s) e.g., a CD4 T cell
  • a CD54-KO PSC- derived T cell e.g., a CD4 T cell, may be resistant to HIV infection.
  • gene editing was employed with different pluripotent stem cells lines, e.g., H9, Hl, PED05, PED04 or derivatives thereof, and the resulting knock down or knock out lines were differentiated, e.g., to cardiomyocytes, endothelial cells or fibroblasts, to show that the cells can differentiate with no apparent defect after knocking down (or knocking out) CD54.
  • cardiomyocytes with a CD54 knockout have a normal karyotype and there are no apparent defects in morphology, e.g., they look to be exactly normal, and they behave similarly, e.g., with regard to contractility and/or differentiation efficiency.
  • the differentiated cells may be tested to determine their immune cell recognition properties.
  • a CD54- knockout PSC-derived T cell may be resistant to HIV infection.
  • any PSC immune cell knockout including T cells (including regulator T cells, effector T cells, and other subsets), B cells, NK cells, monocytes, macrophages, dendritic cells and the like may be prepared and employed in vivo methods.
  • the population of isolated mammalian stem cells have reduced or lack expression of one or more different HLA Class I molecules, one or more different HLA Class II molecules, Beta-2 microglobulin, or have increased expression of CD47, PDL1, secretin, or CTLA4, or any combination thereof or modified versions thereof (e.g., Class II activator (CIITA) or secreted CTLA4-Ig instead of membrane bound CTLA-4).
  • CIITA Class II activator
  • the edits desciebd herein can improve the function of existing gene edits (e.g., by increasing the number of immune cells that become unresponsive to those existing edits/lines, by augmenting the existing edits/lines to improve their immune evasion ability in specific contexts such as when the grafts are vascular in nature, etc.).
  • the method includes isolating, expanding, and/or differentiating, or any combination thereof, the population of hypoimmune mammalian stem cells.
  • the expanded population of hypoimmune mammalian stem cells is differentiated.
  • the isolated cell is differentiated.
  • the population of hypoimmune mammalian stem cells are differentiated to an endothelial cell, cardiac cell, fibroblast cell, pancreatic cell, neural cell, hematopoietic, lymphoid, or islet cell.
  • the differentiated cell is a cardiomyocytc or a neuron.
  • hypoimmune mammalian stem cells e.g., a population of hypoimmune cells, produced by the method.
  • a population of hypoimmune mammalian stem cells have reduced or lack expression of one or more adhesion molecules as a result of genetic modification, e.g., relative to mammalian stem cells or differentiated mammalian cells without the genetic modification, are provided.
  • the genetic modification is to two alleles of a gene encoding an adhesion molecule (a null mutation that is homozygous).
  • the isolated hypoimmune mammalian cells have reduced or lack expression of ICAM-1, ICAM-2, ICAM-3, ICAM-4. ICAM-5, VCAM, MADCAM-1, CD54, P-selectin, E- selectin, L-selectin or a combination thereof.
  • the parent cell is an MHC-1/2 knockout line, thereby providing an additive effect.
  • the parent cell is a hypoimmune mammalian cell that has reduced or lacks expression of CD54, P-selectin, E- selectin, L-selectin, or a combination thereof, which is then modified to knockout MHC1 and/or MHC2.
  • the resulting cells, or the parent overexpress HLA-E and/or CD47 optionally as a result of a genetic modification.
  • the population of hypoimmune mammalian stem cells may be differentiated to endothelial cells, cardiac cells, fibroblasts, pancreatic cells, neural cells, or islet cells.
  • a graft comprising a plurality of one or more hypoimmune differentiated mammalian cell types, wherein the cells are hypoimmune as a result of reduced or a lack of expression of one or more adhesion molecules.
  • the hypoimmune differentiated mammalian cell is a hematopoietic cell, a lymphoid cell, an endothelial cell, cardiac cell, fibroblast cell, pancreatic cell, neural cell, or islet cell.
  • the mammalian cell is a human cell.
  • the graft comprises endothelial cells, fibroblasts and cardiomyocytes.
  • a method of using the graft e.g., by introducing it to a mammal in need thereof.
  • the mammal is a human.
  • the hypoimmune cells are administered as part of a delivery device such as a patch applied to the heart, a bioengineered blood vessel, or an engineered organ which may be comprised of multiple PSC-derived cell types.
  • a method to augment cellular function in a mammal in need thereof comprises administering to the mammal an effective amount of the isolated hypoimmune mammalian stem cells or differentiated cells.
  • the mammal is a human.
  • the mammal is a non-human primate, canine, feline, bovine, equine, swine, ovine, or caprine.
  • the differentiated cells comprise one or more of endothelial cells, cardiomyocytes, fibroblast cells, neurons, hematopoietic cells, lymphoid cells, or islet cells.
  • a method to prevent, inhibit or treat neurological degeneration in a mammal comprises administering to the mammal an effective amount of a composition comprising hypoimmune neural cells that have reduced or lack expression of one or more adhesion molecules as a result of genetic modification.
  • the composition is administered to the central nervous system.
  • the composition is injected.
  • the composition is intracerebroventricularly administered.
  • the mammal is a human.
  • the mammal is a human with Alzhemier’s disease or has had a stroke, and is administered hypoimmune PSC-neurons and/or neural subsets.
  • a method to prevent, inhibit or treat diabetes in a mammal comprises administering to the mammal an effective amount of a composition comprising hypoimmune islet cells that have reduced or lack expression of one or more adhesion molecules as a result of genetic modification.
  • the composition is systemically administered.
  • the composition is injected.
  • the composition is administered to the portal vein.
  • the mammal is a human.
  • the cells are PCS islet cells.
  • the composition comprises a scaffold for the hypoimmune cells which is then implanted in the mammal.
  • a method to prevent, inhibit or treat vascular disease in a mammal includes administering to the mammal an effective amount of a composition comprising hypoimmune endothelial cells that have reduced or lack expression of one or more adhesion molecules as a result of genetic modification.
  • the mammal has peripheral vascular disease.
  • the mammal has, had or is at risk of a myocardial infarction.
  • the composition is injected.
  • the composition is systemically administered.
  • the mammal is a human.
  • the composition is a vascular graft comprising the hypoimmune cells.
  • a method to prevent, inhibit or treat myocardial disease in a mammal comprises administering to the mammal an effective amount of a composition comprising hypoimmune cardiomyocytes that have reduced or lack expression of one or more adhesion molecules as a result of genetic modification.
  • the composition is injected.
  • the composition is administered to the heart.
  • the composition also comprises hypoimmune fibroblasts, hypoimmune endothelial cells, or both.
  • the mammal is a human.
  • the composition is a patch comprising the hypoimmune cells.
  • hypoimmune cells are part of a bioengineered organ or portion thereof, e.g., a decellularized organ or portion thereof is seeded with one or more different types hypoimmune cells.
  • a method to control or increase resistance to viral infection or replication in a mammal in need thereof comprising: administering to the mammal an effective amount of a composition comprising hypoimmune immune cells that have reduced or lack expression of one or more adhesion molecules as a result of genetic modification.
  • the composition is injected.
  • the mammal is a human.
  • the virus is a lentivirus such as HIV.
  • the virus is a rhinovirus or another virus that infects immune cells.
  • the zn vitro method of preparing a population of hypoimmune mammalian stem cells may include the use of a delivery vector.
  • Delivery vectors include, for example, plasmids, viral vectors, liposomes and other lipid-containing complexes, such as lipoplexes (DNA and cationic lipids), polyplexes, e.g., DNA complexed with cationic polymers such as polyethylene glycol, nanoparticles, e.g., magnetic inorganic nanoparticles that bind or are functionalized to bind DNA such as Fc Ari or MnO nanoparticles, microparticles, e.g., formed of poly lactide polygalactide reagents, nanotubes, e.g., silica nanotubes, and other macromolecular complexes capable of mediating delivery of a gene to a host cell.
  • Vectors can also comprise other components or functionalities that further modulate nucleic acid delivery and/or gene expression, or that otherwise provide beneficial properties to the targeted cells.
  • Such other components include, for example, components that influence binding or targeting to cells (including components that mediate cell-type or tissue-specific binding); components that influence uptake of the vector by the cell; components that influence localization of the transferred gene within the cell after uptake (such as agents mediating nuclear localization); and components that influence expression of the gene.
  • Such components also might include markers, such as detectable and/or selectable markers that can be used to detect or select for cells that have taken up and are expressing the nucleic acid delivered by the vector.
  • markers such as detectable and/or selectable markers that can be used to detect or select for cells that have taken up and are expressing the nucleic acid delivered by the vector.
  • a large variety of such vectors are known in the art and are generally available.
  • nucleic acid delivery vehicles deliver guide RNAs, siRNA or vectors for expression thereof, and coding sequences for recombinases including CRISPR, TALENs and zinc finger proteins, or combinations thereof, among others.
  • Other delivery vehicles may include proteins, such as a recombinase or antibody or fragment thereof, or non-protein, non-nucleic acid molecules, optionally in conjunction with nucleic acid.
  • Nucleic acid delivery vectors within the scope of the disclosure include, but are not limited to, isolated nucleic acid, e.g., plasmid-based vectors which may be extrachromosomally maintained, and viral vectors, e.g., recombinant adenovirus, retrovirus, lentivirus, herpesvirus, poxvirus, papilloma virus, or adeno-associated virus, including viral and non-viral vectors which are present in liposomes, e.g., neutral or cationic liposomes, such as DOSPA/DOPE, DOGS/DOPE or DMRIE/DOPE liposomes, and/or associated with other molecules such as DNA-anti-DNA antibody-cationic lipid (DOTMA/DOPE) complexes.
  • isolated nucleic acid e.g., plasmid-based vectors which may be extrachromosomally maintained
  • viral vectors e.g., recombinant adenovirus, retrovirus,
  • Nucleic acid delivery vectors may be administered via any route including, but not limited to, intracranial, intrathecal, intramuscular, buccal, rectal, intravenous or intracoronary administration, and transfer to cells may be enhanced using electroporation and/or iontophoresis, and/or scaffolding such as extracellular matrix or hydrogels, e.g., a hydrogel patch.
  • the CRISPR/Cas System The Type II CRISPR is a well characterized system that carries out targeted DNA double-strand break in four sequential steps. First, two non-coding RNA, the pre-crRNA array and tracrRNA, are transcribed from the CRISPR locus. Second, tracrRNA hybridizes to the repeat regions of the pre-crRNA and mediates the processing of pre- crRNA into mature crRNAs containing individual spacer sequences. Third, the mature crRNA: tracrRNA complex directs Cas9 to the target DNA via Watson-Crick base-pairing between the spacer on the crRNA and the protospacer on the target DNA next to the protospacer adjacent motif (PAM), an additional requirement for target recognition.
  • PAM protospacer adjacent motif
  • Cas9 mediates cleavage of target DNA to create a double- stranded break within the protospacer.
  • Activity of the CRISPR/Cas system comprises of three steps: (i) insertion of alien DNA sequences into the CRISPR array to prevent future attacks, in a process called adaptation, (ii) expression of the relevant proteins, as well as expression and processing of the array, followed by (iii) RNA- mediated interference with the alien nucleic acid.
  • RNA- mediated interference with the alien nucleic acid RNA-mediated interference with the alien nucleic acid.
  • Casl polypeptide refers to CRISPR associated (Cas) protein 1. Cast (COG1518 in the Clusters of Orthologous Group of proteins classification system) is the best marker of the CRISPR-associated systems (CASS). Based on phylogenetic comparisons, seven distinct versions of the CRISPR-associated immune system have been identified (CASS 1-7). Casl polypeptide used in the methods described herein can be any Casl polypeptide present in any prokaryote. In certain embodiments, a Casl polypeptide is a Casl polypeptide of an archaeal microorganism.
  • a Casl polypeptide is a Casl polypeptide of a Euryarchaeota microorganism. In certain embodiments, a Casl polypeptide is a Casl polypeptide of a Crenarchaeota microorganism. In certain embodiments, a Casl polypeptide is a Casl polypeptide of a bacterium. In certain embodiments, a Casl polypeptide is a Casl polypeptide of a gram negative or gram positive bacteria. In certain embodiments, a Cas 1 polypeptide is a Casl polypeptide of Pseudomonas aeruginosa.
  • a Casl polypeptide is a Casl polypeptide of Aquifex aeolicus. In certain embodiments, a Casl polypeptide is a Casl polypeptide that is a member of one of CASsl-7. In certain embodiments, Casl polypeptide is a Casl polypeptide that is a member of CASS3. In certain embodiments, a Casl polypeptide is a Casl polypeptide that is a member of CASS7. In certain embodiments, a Casl polypeptide is a Casl polypeptide that is a member of CASS3 or CASS7.
  • a Casl polypeptide is encoded by a nucleotide sequence provided in GenBank at, e.g., GenelD number: 2781520, 1006874, 9001811, 947228. 3169280, 2650014, 1175302, 3993120, 4380485, 906625, 3165126, 905808, 1454460, 1445886, 1485099, 4274010, 888506, 3169526, 997745, 897836, or 1193018 and/or an amino acid sequence exhibiting homology (e.g., greater than 80%, 90 to 99% including 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%) to the amino acids encoded by these polynucleotides and which polypeptides function as Casl polypeptides.
  • GenBank GenBank at, e.g., GenelD number: 2781520, 1006874, 9001811, 947228. 3169280, 2650014
  • Types I and III both have Cas endonucleases that process the prc-crRNAs, that, when fully processed into crRNAs, assemble a multi-Cas protein complex that is capable of cleaving nucleic acids that are complementary to the crRNA.
  • crRNAs are produced using a different mechanism where a trans-activating RNA (tracrRNA) complementary to repeat sequences in the pre-crRNA, triggers processing by a double strand-specific RNase III in the presence of the Cas9 protein.
  • Cas9 is then able to cleave a target DNA that is complementary to the mature crRNA however cleavage by Cas 9 is dependent both upon base-pairing between the crRNA and the target DNA, and on the presence of a short motif in the crRNA referred to as the PAM sequence (protospacer adjacent motif).
  • the tracrRNA must also be present as it base pairs with the crRNA at its 3' end, and this association triggers Cas9 activity.
  • the Cas9 protein has at least two nuclease domains: one nuclease domain is similar to a HNH endonuclease, while the other resembles a Ruv endonuclease domain.
  • the HNH-type domain appears to be responsible for cleaving the DNA strand that is complementary to the crRNA while the Ruv domain cleaves the non-complementary strand.
  • sgRNA single-guide RNA
  • the engineered tracrRNA:crRNA fusion, or the sgRNA guides Cas9 to cleave the target DNA when a double strand RNA:DNA heterodimer forms between the Cas associated RNAs and the target DNA.
  • This system comprising the Cas9 protein and an engineered sgRNA.
  • Cas polypeptide encompasses a full-length Cas polypeptide, an enzymatically active fragment of a Cas polypeptide, and enzymatically active derivatives of a Cas polypeptide or fragment thereof.
  • Exemplary derivatives of a Cas polypeptide or a fragment thereof include but are not limited to mutants, fusions, covalent modifications of Cas protein or a fragment thereof.
  • RNA Components of CRISPR/Cas The Cas9 related CRISPR/Cas system comprises two RNA non-coding components: tracrRNA and a pre-crRNA array containing nuclease guide sequences (spacers) interspaced by identical direct repeats (DRs). To use a CRISPR/Cas system to accomplish genome engineering, both functions of these RNAs must be present.
  • the tracrRNA and pre-crRNAs are supplied via separate expression constructs or as separate RNAs.
  • a chimeric RNA is constructed where an engineered mature crRNA (conferring target specificity) is fused to a tracrRNA (supplying interaction with the Cas9) to create a chimeric cr-RNA-tracrRNA hybrid (also termed a single guide RNA).
  • Chimeric or sgRNAs can be engineered to comprise a sequence complementary to any desired target.
  • the RNAs comprise 22 bases of complementarity to a target and of the form G[N19], followed by a protospacer-adjacent motif (PAM) of the form NGG.
  • PAM protospacer-adjacent motif
  • sgRNAs can be designed by utilization of a known ZFN target in a gene of interest by (i) aligning the recognition sequence of the ZFN heterodimer with the reference sequence of the relevant genome (human, mouse, or of a particular plant species); (ii) identifying the spacer region between the ZFN half-sites; (iii) identifying the location of the motif G[N20]GG that is closest to the spacer region (when more than one such motif overlaps the spacer, the motif that is centered relative to the spacer is chosen); (iv) using that motif as the core of the sgRNA.
  • This method advantageously relies on proven nuclease targets.
  • sgRNAs can be designed to target any region of interest simply by identifying a suitable target sequence that conforms to the G[N20]GG formula.
  • Donors As noted above, insertion of an exogenous sequence (also called a “donor sequence” or “donor” or “transgene” or “gene of interest”), for example for correction of a mutant gene or for increased expression of a wild-type gene. It will be readily apparent that the donor sequence is typically not identical to the genomic sequence where it is placed. A donor sequence can contain a non-homologous sequence flanked by two regions of homology to allow for efficient HDR at the location of interest. Alternatively, a donor may have no regions of homology to the targeted location in the DNA and may be integrated by NHEI-dependent end joining following cleavage at the target site.
  • donor sequences can comprise a vector molecule containing sequences that are not homologous to the region of interest in cellular chromatin.
  • a donor molecule can contain several, discontinuous regions of homology to cellular chromatin. For example, for targeted insertion of sequences not normally present in a region of interest, said sequences can be present in a donor nucleic acid molecule and flanked by regions of homology to sequence in the region of interest.
  • the donor polynucleotide can be DNA or RNA, single- stranded and/or doublestranded and can be introduced into a cell in linear or circular form. If introduced in linear form, the ends of the donor sequence can be protected (e.g., from exonucleolytic degradation) by methods known to those of skill in the art. For example, one or more didcoxynuclcotidc residues are added to the 3' terminus of a linear molecule and/or self-complementary oligonucleotides are ligated to one or both ends.
  • Additional methods for protecting exogenous polynucleotides from degradation include, but are not limited to, addition of terminal amino group(s) and the use of modified intemucleotide linkages such as, for example, phosphorothioates, phosphoramidates, and O-methyl ribose or deoxyribose residues.
  • a polynucleotide can be introduced into a cell as part of a vector molecule having additional sequences such as, for example, replication origins, promoters and genes encoding antibiotic resistance.
  • donor polynucleotides can be introduced as naked nucleic acid, as nucleic acid complexed with an agent such as a liposome or poloxamer, or can be delivered by viruses (e.g., adenovirus, AAV, herpesvirus, retrovirus, lentivirus and integrase defective lentivirus (IDLV)).
  • viruses e.g., adenovirus, AAV, herpesvirus, retrovirus, lentivirus and integrase defective lentivirus (IDLV)
  • the donor is generally inserted so that its expression is driven by the endogenous promoter at the integration site, namely the promoter that drives expression of the endogenous gene into which the donor is inserted (e.g., highly expressed, albumin, AAVS1, HPRT, etc.).
  • the donor may comprise a promoter and/or enhancer, for example a constitutive promoter or an inducible or tissue specific promoter.
  • the donor molecule may be inserted into an endogenous gene such that all, some or none of the endogenous gene is expressed.
  • a transgene as described herein may be inserted into an albumin or other locus such that some (N-terminal and/or C-terminal to the transgene encoding the lysosomal enzyme) or none of the endogenous albumin sequences are expressed, for example as a fusion with the transgene encoding the lysosomal sequences.
  • the transgene e.g., with or without additional coding sequences such as for albumin
  • is integrated into any endogenous locus for example a safe-harbor locus. See, e.g., U.S. Patent Publication Nos. 2008/0299580; 2008/0159996; and 2010/0218264.
  • endogenous sequences endogenous or part of the transgene
  • the endogenous sequences may be full-length sequences (wild-type or mutant) or partial sequences.
  • the endogenous sequences may be functional.
  • Nonlimiting examples of the function of these full length or partial sequences include increasing the serum half-life of the polypeptide expressed by the transgene (e.g., therapeutic gene) and/or acting as a carrier.
  • exogenous sequences may also include transcriptional or translational regulatory sequences, for example, promoters, enhancers, insulators, internal ribosome entry sites, sequences encoding 2A peptides and/or polyadenylation signals.
  • the genome editing method of the disclosure could involve any other form of genome editing, such as meganucleases, zinc finger nucleases (ZFNs), transcription activator-like effector-based nucleases (TALEN), as well as RNA editing of the precursor mRNAs containing such intronic sequences via CRISPR/Cas 12a, CRISPR/Casl3, or other related genome editing approaches.
  • the agent for altering the target gene is a TALEN system or its equivalent.
  • TALEN Transcriptional Activator-Like Element Nuclease
  • TALE nuclease refers to an artificial nuclease comprising a transcriptional activator like effector DNA binding domain to a DNA cleavage domain, for example, a FokI domain.
  • TALE nucleases can be engineered to target virtually any genomic sequence with high specificity, and that such engineered nucleases can be used in embodiments of the present technology to manipulate the genome of a cell, e.g., by delivering the respective TALEN via a method or strategy disclosed herein under circumstances suitable for the TALEN to bind and cleave its target sequence within the genome of the cell.
  • the delivered TALEN targets a gene or allele associated with a target circRNA.
  • delivery of the TALEN to a subject confers a therapeutic benefit to the subject, such as reducing, eliminating expressing a circRNA in a subject in need thereof.
  • the target gene of a cell, tissue, organ or organism is altered by a nuclease delivered to the cell via a strategy or method disclosed herein, e.g., CRISPR/cas-9, a TALEN, or a zinc-finger nuclease, or a plurality or combination of such nucleases.
  • a single- or double-strand break is introduced at a specific site within the genome by the nuclease, resulting in a disruption of the target genomic sequence, such as an intronic regulatory sequence.
  • Zinc finger refers to a small nucleic acid-binding protein structural motif characterized by a fold and the coordination of one or more zinc ions that stabilize the fold.
  • Zinc fingers encompass a wide variety of differing protein structures. Zinc fingers can be designed to bind a specific sequence of nucleotides, and zinc finger arrays comprising fusions of a series of zinc fingers, can be designed to bind virtually any desired target sequence. Such zinc finger arrays can form a binding domain of a protein, for example, of a nuclease, e.g., if conjugated to a nucleic acid cleavage domain.
  • zinc finger motifs are known to those of skill in the art, including, but not limited to, Cys2His2, Gag knuckle, Treble clef. Zinc ribbon, Zn2/Cys6, and TAZ2 domain-like motifs.
  • a single zinc finger motif binds 3 or 4 nucleotides of a nucleic acid molecule. Accordingly, a zinc finger domain comprising 2 zinc finger motifs may bind 6-8 nucleotides, a zinc finger domain comprising 3 zinc finger motifs may bind 9-12 nucleotides, a zinc finger domain comprising 4 zinc finger motifs may bind 12-16 nucleotides, and so forth.
  • a suitable protein engineering technique can be employed to alter the DNA-binding specificity of zinc fingers and/or design zinc finger fusions to bind virtually any desired target sequence from 3-30 nucleotides in length.
  • Fusions between engineered zinc finger arrays and protein domains that cleave a nucleic acid can be used to generate a “zinc finger nuclease.”
  • a zinc finger nuclease typically comprises a zinc finger domain that binds a specific target site within a nucleic acid molecule, and a nucleic acid cleavage domain that cuts the nucleic acid molecule within or in proximity to the target site bound by the binding domain.
  • Typical engineered zinc finger nucleases comprise a binding domain having between 3 and 6 individual zinc finger motifs and binding target sites ranging from 9 base pairs to 18 base pairs in length. Longer target sites are particularly attractive in situations where it is desired to bind and cleave a target site that is unique in a given genome.
  • the agent for altering gene expression is a zinc finger nuclease or other equivalent.
  • the cleavage domain is the cleavage domain of the type II restriction endonuclease Fokl.
  • Zinc finger nucleases can be designed to target virtually any desired sequence in a given nucleic acid molecule for cleavage, and the possibility to design zinc finger binding domains to bind unique sites in the context of complex genomes allows for targeted cleavage of a single genomic site in living cells, for example, to achieve a targeted genomic alteration of therapeutic value.
  • Targeting a double-strand break to a desired genomic locus can be used to introduce frame-shift mutations into the coding sequence of a gene due to the error-prone nature of the non-homologous DNA repair pathway.
  • Zinc finger nucleases can be generated to target a site of interest by methods well known to those of skill in the art. For example, zinc finger binding domains with a desired specificity can be designed by combining individual zinc finger motifs of known specificity. The structure of the zinc finger protein Zif268 bound to DNA has informed much of the work in this field and the concept of obtaining zinc fingers for each of the 64 possible base pair triplets and then mixing and matching these modular zinc fingers to design proteins with any desired sequence specificity has been described.
  • separate zinc fingers that each recognize a 3 base pair DNA sequence are combined to generate 3-, 4-, 5-, or 6-finger arrays that recognize target sites ranging from 9 base pairs to 18 base pairs in length. In some embodiments, longer arrays are contemplated. In other embodiments, 2-finger modules recognizing 6-8 nucleotides are combined to generate 4-, 6-, or 8-zinc finger arrays. In some embodiments, bacterial or phage display is employed to develop a zinc finger domain that recognizes a desired nucleic acid sequence, for example, a desired nuclease target site of 3-30 bp in length.
  • Zinc finger nucleases in some embodiments, comprise a zinc finger binding domain and a cleavage domain fused or otherwise conjugated to each other via a linker, for example, a polypeptide linker.
  • the length of the linker determines the distance of the cut from the nucleic acid sequence bound by the zinc finger domain. If a shorter linker is used, the cleavage domain will cut the nucleic acid closer to the bound nucleic acid sequence, while a longer linker will result in a greater distance between the cut and the bound nucleic acid sequence.
  • the cleavage domain of a zinc finger nuclease has to dimerize in order to cut a bound nucleic acid.
  • the dimer is a heterodimer of two monomers, each of which comprise a different zinc finger binding domain.
  • the dimer may comprise one monomer comprising zinc finger domain A conjugated to a FokI cleavage domain, and one monomer comprising zinc finger domain B conjugated to a FokI cleavage domain.
  • zinc finger domain A binds a nucleic acid sequence on one side of the target site
  • zinc finger domain B binds a nucleic acid sequence on the other side of the target site
  • the dimerize FokI domain cuts the nucleic acid in between the zinc finger domain binding sites.
  • siRNA delivery vectors within the scope of the disclosure include, but are not limited to, isolated nucleic acid, e.g., plasmid-based vectors which may be extrachromosomally maintained, and viral vectors, e.g., recombinant adenovirus, retrovirus, lentivirus, herpesvirus, poxvirus, papilloma virus, or adeno-associated virus, including viral and non-viral vectors which are present in liposomes, e.g., neutral or cationic liposomes, such as DOSPA/DOPE, DOGS/DOPE or DMRIE/DOPE liposomes, and/or associated with other molecules such as DNA-anti-DNA antibody-cationic lipid (DOTMA/DOPE) complexes or natural or synthetic polymers.
  • viral vectors e.g., recombinant adenovirus, retrovirus, lentivirus, herpesvirus, poxvirus, papilloma virus, or adeno
  • Nucleic acid delivery vectors may be administered via any route including, but not limited to, intracranial, intrathecal, intramuscular, buccal, rectal, intravenous or intracoronary administration, and transfer to cells may be enhanced using electroporation and/or iontophoresis, and/or scaffolding such as extracellular matrix or hydrogels, e.g., a hydrogel patch.
  • the vector is a viral vector.
  • viral vectors include, for example, retroviral vectors, lentivirus vectors, herpes simplex virus (HSV)-based vectors, parvovirus-based vectors, e.g., adeno-associated virus (AAV)-based vectors, AAV-adenoviral chimeric vectors, and adenovirus-based vectors.
  • HSV herpes simplex virus
  • AAV adeno-associated virus
  • AAV-adenoviral chimeric vectors e.g., AAV-adenoviral chimeric vectors
  • adenovirus-based vectors e.g., adeno-associated virus (AAV)-based vectors.
  • Retroviral vectors exhibit several distinctive features including their ability to stably and precisely integrate into the host genome providing long-term transgene expression. These vectors can be manipulated ex vivo to eliminate infectious gene particles to minimize the risk of systemic infection and patient-to-patient transmission. Pseudotyped retroviral vectors can alter host cell tropism.
  • Lentiviruses are derived from a family of retroviruses that include human immunodeficiency virus and feline immunodeficiency virus. However, unlike retroviruses that only infect dividing cells, lentiviruses can infect both dividing and nondividing cells. Although lentiviruses have specific tropisms, pseudotyping the viral envelope with vesicular stomatitis virus yields virus with a broader range.
  • Adenoviral vectors may be rendered replication-incompetent by deleting the early (E1A and E1B) genes responsible for viral gene expression from the genome and are stably maintained into the host cells in an extrachromosomal form. These vectors have the ability to transfect both replicating and nonreplicating cells. Adenoviral vectors have been shown to result in transient expression of therapeutic genes in vivo, peaking at 7 days and lasting approximately 4 weeks. In addition, adenoviral vectors can be produced at very high titers, allowing efficient gene therapy with small volumes of virus.
  • Adeno-associated virus vectors Recombinant adeno-associated viruses (rAAV) are derived from nonpathogenic parvoviruses, evoke essentially no cellular immune response, and produce transgene expression lasting months in most systems. Moreover, like adenovirus, adeno-associated virus vectors also have the capability to infect replicating and nonreplicating cells.
  • AAV vectors include but are not limited to AAV1, AAV2, AAV5. AAV7, AAV8, AAV9 or AAVrhlO, including chimeric viruses where the AAV genome is from a different source than the capsid.
  • Plasmid DNA vectors Plasmid DNA is often referred to as “naked DNA” to indicate the absence of a more elaborate packaging system. Direct injection of plasmid DNA to myocardial cells in vivo has been accomplished. Plasmid-based vectors are relatively nonimmunogenic and nonpathogenic, with the potential to stably integrate in the cellular genome, resulting in long-term gene expression in postmitotic cells in vivo. Furthermore, plasmid DNA is rapidly degraded in the blood stream; therefore, the chance of transgene expression in distant organ systems is negligible. Plasmid DNA may be delivered to cells as part of a macromolecular complex, e.g., a liposome or DNA-protein complex, and delivery may be enhanced using techniques including electroporation.
  • a macromolecular complex e.g., a liposome or DNA-protein complex
  • hypoimmune Cells One hurdle preventing widespread use of differentiated stem cells as therapeutics is immune rejection of the transplanted cells.
  • the present disclosure describes a platform strategy for modifying stem cells such as pluripotent stem cell lines prior to cell therapy manufacturing.
  • the resulting cells, including cell differentiated therefrom, do not disrupt the immune system balance needed to prevent transplant rejection while also preventing tumor development, and so can be used in biomanufacturing differentiated stem cell therapies.
  • gene-editing approaches may allow for immune evasion of stem cells used in therapies, due to the nature of stem cells such as iPSCs (clonal cell lines, massively scalable).
  • the present disclosure provides for an alternative mechanism of evading the immune response. Further, the present approach may diminish the prospects of graft loss via two separate but complementary mechanisms by which grafts are rejected, direct immune cell contact and indirect inflammatory processes.
  • PSC such as iPSC or ESC may be gene edited or otherwise modified, e.g., genetically, e.g., in GMO facilities. Banks of cells are made that are suitable for clinical use.
  • CD54 knock out (CD54-KO) stem cells that optionally include additional gene edits such as B2M KO) are differentiated into cell therapies (e.g., cardiomyocytes for myocardial infarction, endothelial cells for vascular disease, neurons for dementia, pancreatic islet cells for diabetes, hepatocytes for liver disease, T cells for HIV infection, retinal pigment epithelial cells for macular degeneration, and the like).
  • These cell therapies can be banked and used as an as-needed cell therapy.
  • the cells are transplanted into patients, e.g., as a therapeutic, curative and/or immune-tolerated cell therapy.
  • these patients may not need to take immunosuppressive drugs (which have a number of non-trivial adverse effects) or may require lesser amounts of immunosuppressive drugs because the cell therapy itself is protected from rejection obviating the need for systemic and/or multi-drug immune-suppression.
  • therapies such as a cardiac therapy can save lives as well as improve the quality-of-life for millions of patients.
  • Exemplary Proteins for Knock-down/Knock-out The disclosure provides for cells having decreased expression of one or more adhesion molecules.
  • Adhesion molecules are generally divided into five groups: integrins, selectins, cadherins, members of the immunoglobulin superfamily (IgSF) including ncctins and others such as mucins.
  • IgSF immunoglobulin superfamily
  • Integrins typically bind to the extracellular matrix, while selectins, cadherins, and IgSF members are associated with cell-cell adhesion.
  • P-, E- and L-selectins originally based on which cell types they were found in: platelets, endothelial cells and leukocytes.
  • IgSF immunoglobulin or immunoglobulin-like domain and most members are type I transmembrane proteins with an extracellular domain (containing the Ig domain[s]), transmembrane domain and a cytoplasmic tail.
  • MHC major histocompatibility complex
  • TCR T cell receptor
  • IAMs Intercellular adhesion molecules
  • VCAMs vascular cell adhesion molecules
  • MAdCAM-1 activated leukocyte cell adhesion molecule
  • ALCAM activated leukocyte cell adhesion molecule
  • Integrins are large heterodimers consisting of a- and P-chains that together form the intact receptor in the plasma membrane.
  • the stem cells have reduced or lack of expression of human ICAM-1 (CD54), e.g., a polypeptide having
  • the stem cells have reduced or lack of expression of human
  • CD62 P-selectin
  • the stem cells have reduced or lack of expression of human
  • CD62E E-selectin
  • compositions of the present disclosure suitable for inoculation, e.g., nasal, parenteral or oral administration, such as by intravenous, intramuscular, intranasal, topical or subcutaneous routes, comprise one or more hypoimmune cell types, optionally further comprising sterile aqueous or non-aqueous solutions, suspensions, and emulsions.
  • the compositions can further comprise auxiliary agents or excipients, as known in the art.
  • the composition is generally presented in the form of individual doses (unit doses).
  • Preparations for parenteral administration include sterile aqueous or nonaqueous solutions, suspensions, and/or emulsions, which may contain auxiliary agents or excipients known in the art.
  • non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate.
  • Carriers or occlusive dressings can be used to increase skin permeability and enhance antigen absorption.
  • Liquid dosage forms for oral administration may generally comprise a liposome solution containing the liquid dosage form.
  • Suitable forms for suspending liposomes include emulsions, suspensions, solutions, syrups, and elixirs containing inert diluents commonly used in the art, such as purified water. Besides the inert diluents, such compositions can also include adjuvants, wetting agents, emulsifying and suspending agents, or sweetening, flavoring, or perfuming agents.
  • the composition is a patch.
  • a cardiac patch may include skeletal myoblasts, cardiac stem/stromal cells, mesenchymal stem cells (MSCs), and/or human pluripotent stem cells, which have a gene knockdown or knockout.
  • the patch may be formed of synthetic or natural components, e.g., polymers. Synthetic materials include but are not limited to a polymer such as poly(vinyl alcohol) (PVA), poly(lactic-co-glycolic) acid (PLGA), poly-(L- lactic) acid (PLLA) or polyurethanes (PU).
  • PVA poly(vinyl alcohol)
  • PLGA poly(lactic-co-glycolic) acid
  • PLLA poly-(L- lactic) acid
  • PU polyurethanes
  • the patch may include a hydrogel. Different natural materials may also be employed such as collagen, fibrin, alginate, hyaluronic acid, gelatin, and decellularized extra-cellular matrix (ECM).
  • compositions when used for administration to an individual, it can further comprise salts, buffers, adjuvants, or other substances which are desirable for maintaining or improving the efficacy of the composition.
  • the pharmaceutical compositions comprise a therapeutically effective amount of the cells, and a pharmaceutically acceptable carrier.
  • pharmaceutically acceptable means approved by a regulatory agency of the Federal or a state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeiae for use in animals, and more particularly in humans.
  • carrier refers to a diluent, adjuvant, excipient, or vehicle with which the pharmaceutical composition is administered. Saline solutions and aqueous dextrose and glycerol solutions can also be employed as liquid carriers, particularly for injectable solutions.
  • Exemplary pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol and the like. These compositions can take the form of solutions, suspensions, emulsion, tablets, pills, capsules, powders, sustained-release formulations and the like. These compositions can be formulated as a suppository. Oral formulation can include standard carriers such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate, etc.
  • compositions will contain a therapeutically effective amount of the virus, e.g., in purified form, together with a suitable amount of carrier so as to provide the form for proper administration to the patient.
  • suitable pharmaceutical carriers are described in “Remington's Pharmaceutical Sciences” by E. W. Martin.
  • Such compositions will contain a therapeutically effective amount of the virus, e.g., in purified form, together with a suitable amount of carrier so as to provide the form for proper administration to the patient.
  • the formulation should suit the mode of administration.
  • compositions may be systemically administered, e.g., orally, locally administered, e.g., to an organ or intramuscularly, in combination with a pharmaceutically acceptable vehicle such as an inert diluent.
  • a pharmaceutically acceptable vehicle such as an inert diluent.
  • the cells may be combined with one or more excipients and used in the form of ingestible capsules, elixirs, suspensions, syrups, wafers, and the like.
  • Such compositions should contain at least 0.1% of active compound.
  • the percentage of the compositions and preparations may, of course, be varied and may conveniently be between about 2 to about 60% of the weight of a given unit dosage form. The amount of active compound in such useful compositions is such that an effective dosage level will be obtained.
  • composition also be administered intravenously or intraperitoneally by infusion or injection.
  • Solutions of the cells can be prepared in water or a suitable buffer, optionally mixed with a nontoxic surfactant.
  • Dispersions can also be prepared in glycerol, liquid polyethylene glycols, triacetin, and mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of undesirable microorganisms.
  • the pharmaceutical dosage forms suitable for injection or infusion can include sterile aqueous solutions or dispersions or sterile powders comprising the active ingredient which are adapted for the extemporaneous preparation of sterile injectable or infusible solutions or dispersions, optionally encapsulated in liposomes.
  • the liquid carrier or vehicle can be a solvent or liquid dispersion medium comprising, for example, water, ethanol, a polyol (for example, glycerol, propylene glycol, liquid polyethylene glycols, and the like), vegetable oils, nontoxic glyceryl esters, and suitable mixtures thereof.
  • the fluidity can be maintained, for example, by the formation of liposomes, by the maintenance of the particle size in the case of dispersions or by the use of surfactants.
  • the prevention of the action of undesirable microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like.
  • isotonic agents for example, sugars, buffers or sodium chloride.
  • Sterile injectable solutions are prepared by incorporating the cells in an amount in the appropriate solvent with various of the other ingredients enumerated above, if required, followed by filter sterilization.
  • Useful liquid carriers include water, alcohols or glycols or water- alcohol/gly col blends, in which the present viruses can be dissolved or dispersed at effective levels, optionally with the aid of non-toxic surfactants.
  • Adjuvants such as fragrances and additional antimicrobial agents can be added to optimize the properties for a given use.
  • the resultant liquid compositions can be applied from absorbent pads, used to impregnate bandages and other dressings, or sprayed onto the affected area using pump-type or aerosol sprayers.
  • compositions may be for either a “prophylactic” or “therapeutic” purpose.
  • the compositions are provided before any symptom or clinical sign of a disease or disorder becomes manifest.
  • the prophylactic administration of the composition serves to prevent or attenuate any subsequent disease or disorder or symptom thereof.
  • the compositions are provided before any symptom or clinical sign of a disease becomes manifest.
  • the prophylactic administration of the composition serves to prevent or attenuate one or more symptoms or clinical signs associated with the disease.
  • the cells are provided upon the detection of a symptom or clinical sign of a disease or disorder.
  • the therapeutic administration of the cell(s) serves to attenuate one or more symptoms of the disease or disorder.
  • a composition comprising cells is provided upon the detection of a symptom or clinical sign of the disease.
  • the therapeutic administration of the cells serves to attenuate a symptom or clinical sign of that disease.
  • composition of the present disclosure may be provided either before the onset of disease (so as to prevent or attenuate the disease) or after detection of a disease.
  • the composition may be provided before any symptom or clinical sign of a disorder or disease is manifested or after one or more symptoms are detected.
  • a composition is said to be “pharmacologically acceptable” if its administration can be tolerated by a recipient mammal. Such an agent is said to be administered in a “therapeutically effective amount” if the amount administered is physiologically significant.
  • a composition is physiologically significant if its presence results in a detectable change in the physiology of a recipient patient, e.g., enhances at least one primary or secondary humoral or cellular immune response against at least one strain of a virus.
  • the “protection” provided need not be absolute, i.e., the disease or a symptom thereof need not be totally prevented or eradicated, if there is a statistically significant improvement compared with a control population or set of mammals. Protection may be limited to mitigating the severity or rapidity of onset of symptoms or clinical signs of the disease. For example, a cardiac patch may improve cardiac ejection fraction, thereby allowing for patients to move/exercise better, which in turn enhances their quality of life.
  • composition having cells is said to prevent or attenuate a disease if its administration results either in the total or partial attenuation (i.e., suppression) of a clinical sign or condition of the disease.
  • a cell type may be administered by any means that achieve the intended purposes.
  • administration of such a composition may be by various parenteral routes such as subcutaneous, intravenous, intradermal, intramuscular, intraperitoneal, intranasal, oral or transdermal routes.
  • Parenteral administration can be accomplished by bolus injection or by gradual perfusion over time.
  • the cells are part of a substrate, e.g., patch or hydrogel.
  • the cells may be seeded onto or embedded in a substrate.
  • the substrate is a synthetic graft, e.g., a blood vessel graft or intestinal graft.
  • the cells are part of an organ or a portion thereof.
  • the cells may be seeded onto a dcccllularizcd organ or portion thereof.
  • An exemplary regimen for preventing, suppressing, or treating a pathology comprises administration of an effective amount of a composition as described herein, administered as a single treatment, or repeated dosages, for instance, over a period up to and including between one week and about 10 or more years, or any range or value therein.
  • an “effective amount” of a composition is one that is sufficient to achieve a desired effect. It is understood that the effective dosage may be dependent upon the species, age, sex, health, and weight of the recipient, kind of concurrent treatment, if any, frequency of treatment, and the nature of the effect wanted. The ranges of effective doses provided below arc not intended to limit dose ranges.
  • Exemplary doses include but are not limited to from about 10 4 to 10 8 cells, 10 6 to 10 8 cells, 10 6 to 10 10 cells, or 10 8 to 10 12 cells, or more, or from about 10 6 to 10 8 cells, 10 8 to 10 10 cells, or 10 10 to 10 12 cells; from about 10 4 to 10 8 cells/kg, 10 6 to 10 8 cells/kg, 10 6 to 10 10 cells/kg, or 10 8 to 10 12 cells/kg, or more, or from about 10 6 to 10 8 cells/kg, 10 8 to 10 10 cells, or 10 10 to 10 12 cells/kg.
  • a “vector” refers to a macromolecule or association of macromolecules that comprises or associates with a polynucleotide, and which can be used to mediate delivery of the polynucleotide to a cell, either in vitro or in vivo.
  • Illustrative vectors include, for example, plasmids, viral vectors, liposomes and other nucleic acid delivery vehicles.
  • the polynucleotide to be delivered may comprise a coding sequence of interest in gene therapy (such as a gene encoding a protein of therapeutic interest), a coding sequence of interest in vaccine development (such as a polynucleotide expressing a protein, polypeptide or peptide suitable for eliciting an immune response in a mammal), and/or a selectable or detectable marker.
  • Transduction is terms referring to a process for the introduction of an exogenous polynucleotide into a host cell leading to expression of the polynucleotide, e.g., the transgene in the cell, and includes the use of recombinant virus to introduce the exogenous polynucleotide to the host cell.
  • Transduction, transfection or transformation of a polynucleotide in a cell may be determined by methods well known to the art including, but not limited to, protein expression (including steady state levels), c.g., by ELISA, flow cytometry and Western blot, measurement of DNA and RNA by hybridization assays, e.g., Northern blots, Southern blots and gel shift mobility assays.
  • Methods used for the introduction of the exogenous polynucleotide include well-known techniques such as viral infection or transfection, lipofection, transformation and electroporation, as well as other non-viral gene delivery techniques.
  • the introduced polynucleotide may be stably or transiently maintained in the host cell.
  • Gene delivery refers to the introduction of an exogenous polynucleotide into a cell for gene transfer, and may encompass targeting, binding, uptake, transport, localization, replicon integration and expression.
  • Gene transfer refers to the introduction of an exogenous polynucleotide into a cell which may encompass targeting, binding, uptake, transport, localization and replicon integration, but is distinct from and does not imply subsequent expression of the gene.
  • Gene expression or “expression” refers to the process of gene transcription, translation, and post-translational modification.
  • infectious virus or viral particle is one that comprises a polynucleotide component which it is capable of delivering into a cell for which the viral species is trophic.
  • the term does not necessarily imply any replication capacity of the virus.
  • nucleic acid refers to a deoxyribonucleotide or ribonucleotide polymer, in linear or circular conformation, and in either single-or double-stranded form.
  • polynucleotide refers to a deoxyribonucleotide or ribonucleotide polymer, in linear or circular conformation, and in either single-or double-stranded form.
  • these terms are not to be construed as limiting with respect to the length of a polymer.
  • the terms can encompass known analogues of natural nucleotides, as well as nucleotides that are modified in the base, sugar and/or phosphate moieties (e.g., phosphorothioate backbones).
  • an analogue of a particular nucleotide has the same base-pairing specificity, i.e., an analog of A will base-pair with T.
  • an “isolated” polynucleotide e.g., plasmid, virus, polypeptide, cell, or other substance refers to a preparation of the substance devoid of at least some of the other components that may also be present where the substance or a similar substance naturally occurs or is initially prepared from. Thus, for example, an isolated substance may be prepared by using a purification technique to enrich it from a source mixture. Isolated nucleic acid, peptide or polypeptide is present in a form or setting that is different from that in which it is found in nature.
  • a given DNA sequence (c.g., a gene) is found on the host cell chromosome in proximity to neighboring genes; RNA sequences, such as a specific mRNA sequence encoding a specific protein, are found in the cell as a mixture with numerous other mRNAs that encode a multitude of proteins.
  • the isolated nucleic acid molecule may be present in single-stranded or double-stranded form. When an isolated nucleic acid molecule is to be utilized to express a protein, the molecule will contain at a minimum the sense or coding strand (i.e., the molecule may single-stranded), but may contain both the sense and anti-sense strands (i.e., the molecule may be double-stranded).
  • Enrichment can be measured on an absolute basis, such as weight per volume of solution, or it can be measured in relation to a second, potentially interfering substance present in the source mixture. Increasing enrichments of the embodiments of this invention are envisioned. Thus, for example, a 2-fold enrichment, 10-fold enrichment, 100-fold enrichment, or a 1000-fold enrichment.
  • a “transcriptional regulatory sequence” refers to a genomic region that controls the transcription of a gene or coding sequence to which it is operably linked.
  • Transcriptional regulatory sequences of use in the present invention generally include at least one transcriptional promoter and may also include one or more enhancers and/or terminators of transcription.
  • “Operably linked” refers to an arrangement of two or more components, wherein the components so described are in a relationship permitting them to function in a coordinated manner.
  • a transcriptional regulatory sequence or a promoter is operably linked to a coding sequence if the TRS or promoter promotes transcription of the coding sequence.
  • An operably linked TRS is generally joined in cis with the coding sequence, but it is not necessarily directly adjacent to it.
  • operative linkage and “operatively linked” (or “operably linked”) are used interchangeably with reference to a juxtaposition of two or more components (such as sequence elements), in which the components are arranged such that both components function normally and allow the possibility that at least one of the components can mediate a function that is exerted upon at least one of the other components.
  • a transcriptional regulatory sequence such as a promoter
  • a transcriptional regulatory sequence is generally operatively linked in cis with a coding sequence, but need not be directly adjacent to it.
  • an enhancer is a transcriptional regulatory sequence that is operatively linked to a coding sequence.
  • Heterologous means derived from a genotypically distinct entity from the entity to which it is compared.
  • a polynucleotide introduced by genetic engineering techniques into a different cell type is a heterologous polynucleotide (and, when expressed, can encode a heterologous polypeptide).
  • a transcriptional regulatory element such as a promoter that is removed from its native coding sequence and operably linked to a different coding sequence is a heterologous transcriptional regulatory element.
  • a “terminator” refers to a polynucleotide sequence that tends to diminish or prevent read-through transcription (i.e., it diminishes or prevent transcription originating on one side of the terminator from continuing through to the other side of the terminator).
  • the degree to which transcription is disrupted is typically a function of the base sequence and/or the length of the terminator sequence.
  • transcriptional termination sequences are specific sequences that tend to disrupt read-through transcription by RNA polymerase, presumably by causing the RNA polymerase molecule to stop and/or disengage from the DNA being transcribed.
  • sequence-specific terminators include polyadenylation (“poly A”) sequences, e.g., SV40 polyA.
  • poly A polyadenylation
  • insertions of relatively long DNA sequences between a promoter and a coding region also tend to disrupt transcription of the coding region, generally in proportion to the length of the intervening sequence. This effect presumably arises because there is always some tendency for an RNA polymerase molecule to become disengaged from the DNA being transcribed, and increasing the length of the sequence to be traversed before reaching the coding region would generally increase the likelihood that disengagement would occur before transcription of the coding region was completed or possibly even initiated.
  • Terminators may thus prevent transcription from only one direction (“uni-directional” terminators) or from both directions (“bi-directional” terminators), and may be comprised of sequence-specific termination sequences or sequence-non-specific terminators or both.
  • a variety of such terminator sequences are known in the ail; and illustrative uses of such sequences within the context of the present invention are provided below.
  • “Host cells,” “cell lines,” “cell cultures,” “packaging cell line” and other such terms denote higher eukaryotic cells, such as mammalian cells including human cells, useful in the present disclosure. These cells include the progeny of the original cell that was transduced. It is understood that the progeny of a single cell may not necessarily be completely identical (in morphology or in genomic complement) to the original parent cell.
  • a recombinant virus is a viral particle comprising a recombinant polynucleotide. The terms respectively include replicates of the original polynucleotide construct and progeny of the original virus construct.
  • control element or “control sequence” is a nucleotide sequence involved in an interaction of molecules that contributes to the functional regulation of a polynucleotide, including replication, duplication, transcription, splicing, translation, or degradation of the polynucleotide. The regulation may affect the frequency, speed, or specificity of the process, and may be enhancing or inhibitory in nature.
  • Control elements known in the art include, for example, transcriptional regulatory sequences such as promoters and enhancers.
  • a promoter is a DNA region capable under certain conditions of binding RNA polymerase and initiating transcription of a coding region usually located downstream (in the 3' direction) from the promoter. Promoters include AAV promoters, e.g., P5, P19, P40 and AAV ITR promoters, as well as heterologous promoters.
  • An “expression vector” is a vector comprising a region which encodes a gene product of interest, and is used for effecting the expression of the gene product in an intended target cell.
  • An expression vector also comprises control elements operatively linked to the encoding region to facilitate expression of the protein in the target.
  • the combination of control elements and a gene or genes to which they are operably linked for expression is sometimes referred to as an “expression cassette,” a large number of which are known and available in the art or can be readily constructed from components that are available in the art.
  • polypeptide and protein are used interchangeably herein to refer to polymers of amino acids of any length.
  • the terms also encompass an amino acid polymer that has been modified; for example, disulfide bond formation, glycosylation, acetylation, phosphorylation, lipidation, or conjugation with a labeling component.
  • exogenous when used in relation to a protein, gene, nucleic acid, or polynucleotide in a cell or organism refers to a protein, gene, nucleic acid, or polynucleotide which has been introduced into the cell or organism by artificial or natural means.
  • An exogenous nucleic acid may be from a different organism or cell, or it may be one or more additional copies of a nucleic acid which occurs naturally within the organism or cell.
  • an exogenous nucleic acid is in a chromosomal location different from that of natural cells, or is otherwise flanked by a different nucleic acid sequence than that found in nature, e.g., an expression cassette which links a promoter from one gene to an open reading frame for a gene product from a different gene.
  • an “exogenous” molecule is a molecule that is not normally present in a cell, but can be introduced into a cell by one or more genetic, biochemical or other methods. “Normal presence in the cell” is determined with respect to the particular developmental stage and environmental conditions of the cell. Thus, for example, a molecule that is present only during embryonic development of muscle is an exogenous molecule with respect to an adult muscle cell. Similarly, a molecule induced by heat shock is an exogenous molecule with respect to a non-heat- shocked cell.
  • An exogenous molecule can comprise, for example, a functioning version of a malfunctioning endogenous molecule or a malfunctioning version of a normally- functioning endogenous molecule.
  • An exogenous molecule can be, among other things, a small molecule, such as is generated by a combinatorial chemistry process, or a macromolecule such as a protein, nucleic acid, carbohydrate, lipid, glycoprotein, lipoprotein, polysaccharide, any modified derivative of the above molecules, or any complex comprising one or more of the above molecules.
  • Nucleic acids include DNA and RNA, can be single-or double- stranded; can be linear, branched or circular; and can be of any length. Nucleic acids include those capable of forming duplexes, as well as triplex-forming nucleic acids.
  • an exogenous molecule can be the same type of molecule as an endogenous molecule, e.g., an exogenous protein or nucleic acid.
  • an exogenous nucleic acid can comprise an infecting viral genome, a plasmid or episome introduced into a cell, or a chromosome that is not normally present in the cell.
  • exogenous molecules include, but are not limited to, lipid-mcdiatcd transfer (c.g., liposomes, including neutral and cationic lipids), electroporation, direct injection, cell fusion, particle bombardment, calcium phosphate co-precipitation, DEAE- dextran-mediated transfer and viral vector-mediated transfer.
  • An exogenous molecule can also be the same type of molecule as an endogenous molecule but derived from a different species than the cell is derived from.
  • a human nucleic acid sequence may be introduced into a cell line originally derived from a mouse or hamster.
  • An exogenous molecule can be, among other things, a small molecule, such as is generated by a combinatorial chemistry process, or a macromolecule such as a protein, nucleic acid, carbohydrate, lipid, glycoprotein, lipoprotein, polysaccharide, any modified derivative of the above molecules, or any complex comprising one or more of the above molecules.
  • Nucleic acids include DNA and RNA, can be single-or double- stranded; can be linear, branched or circular; and can be of any length. Nucleic acids include those capable of forming duplexes, as well as triplex-forming nucleic acids.
  • an “endogenous” molecule is one that is normally present in a particular cell at a particular developmental stage under particular environmental conditions.
  • an endogenous nucleic acid can comprise a chromosome, the genome of a mitochondrion, chloroplast or other organelle, or a naturally-occurring episomal nucleic acid.
  • Transformed or “transgenic” is used herein to include any host cell or cell line, which has been altered or augmented by the presence of at least one recombinant DNA sequence.
  • the host cells of the present invention are typically produced by transfection with a DNA sequence in a plasmid expression vector, as an isolated linear- DNA sequence, or infection with a recombinant viral vector.
  • sequence homology means the proportion of base matches between two nucleic acid sequences or the proportion amino acid matches between two amino acid sequences. When sequence homology is expressed as a percentage, e.g., 50%, the percentage denotes the proportion of matches over the length of a selected sequence that is compared to some other sequence. Gaps (in either of the two sequences) are permitted to maximize matching; gap lengths of 15 bases or less are usually used, 6 bases or less e.g., with 2 bases or less.
  • the sequence homology between the target nucleic acid and the oligonucleotide sequence is generally not less than 17 target base matches out of 20 possible oligonucleotide base pair matches (85%); not less than 9 matches out of 10 possible base pair matches (90%), or not less than 19 matches out of 20 possible base pair matches (95%).
  • Two amino acid sequences are homologous if there is a partial or complete identity between their sequences. For example, 85% homology means that 85% of the amino acids are identical when the two sequences are aligned for maximum matching. Gaps (in either of the two sequences being matched) are allowed in maximizing matching; gap lengths of 5 or less or with 2 or less.
  • two protein sequences are homologous, as this term is used herein, if they have an alignment score of at more than 5 (in standard deviation units) using the program ALIGN with the mutation data matrix and a gap penalty of 6 or greater.
  • the two sequences or parts thereof are more homologous if their amino acids are greater than or equal to 50% identical when optimally aligned using the ALIGN program.
  • the term “corresponds to” is used herein to mean that a polynucleotide sequence is structurally related to all or a portion of a reference polynucleotide sequence, or that a polypeptide sequence is structurally related to all or a portion of a reference polypeptide sequence, e.g., they have at least 80%, 85%, 90%, 95% or more, e.g., 99% or 100%, sequence identity.
  • the term “complementary to” is used herein to mean that the complementary sequence is homologous to all or a portion of a reference polynucleotide sequence. For illustration, the nucleotide sequence “TAT AC” corresponds to a reference sequence “TAT AC” and is complementary to a reference sequence “GT AT A”.
  • sequence identity means that two polynucleotide sequences are identical (i.e., on a nucleotide -by-nucleotide basis) over the window of comparison.
  • percentage of sequence identity means that two polynucleotide sequences are identical (i.e., on a nucleotide-by-nucleotide basis) over the window of comparison.
  • percentage of sequence identity is calculated by comparing two optimally aligned sequences over the window of comparison, determining the number of positions at which the identical nucleic acid base (e.g., A, T, C, G, U, or I) occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison (i.e., the window size), and multiplying the result by 100 to yield the percentage of sequence identity.
  • the identical nucleic acid base e.g., A, T, C, G, U, or I
  • substantially identical denote a characteristic of a polynucleotide sequence, wherein the polynucleotide comprises a sequence that has at least 85 percent sequence identity, e.g., at least 90 to 95 percent sequence identity, or at least 99 percent sequence identity as compared to a reference sequence over a comparison window of at least 20 nucleotide positions, frequently over a window of at least 20-50 nucleotides, wherein the percentage of sequence identity is calculated by comparing the reference sequence to the polynucleotide sequence which may include deletions or additions which total 20 percent or less of the reference sequence over the window of comparison.
  • Constant amino acid substitutions are, for example, aspartic-glutamic as polar acidic amino acids; lysine/arginine/histidine as polar basic amino acids; leucine/isoleucine/methionine/valine/alanine/glycine/proline as non-polar or hydrophobic amino acids; serine/ threonine as polar or uncharged hydrophilic amino acids.
  • Conservative amino acid substitution also includes groupings based on side chains.
  • a group of amino acids having aliphatic side chains is glycine, alanine, valine, leucine, and isoleucine; a group of amino acids having aliphatic-hydroxyl side chains is serine and threonine; a group of amino acids having amide-containing side chains is asparagine and glutamine; a group of amino acids having aromatic side chains is phenylalanine, tyrosine, and tryptophan; a group of amino acids having basic side chains is lysine, arginine, and histidine; and a group of amino acids having sulfur- containing side chains is cysteine and methionine.
  • Naturally occurring residues are divided into groups based on common side-chain properties: (1) hydrophobic: norleucine, met, ala, val, leu, ile; (2) neutral hydrophilic: cys, ser, thr; (3) acidic: asp, glu; (4) basic: asn, gin, his, lys, arg; (5) residues that influence chain orientation: gly, pro; and (6) aromatic; trp, tyr, phe.
  • Nonconservative substitutions entail exchanging a member of one of the classes described above for another.
  • mammals include, for example, humans; non-human primates, e.g., apes and monkeys; and non- primates, e.g., dogs, cats, rats, mice, cattle, horses, sheep, and goats.
  • Non-mammals include, for example, fish and birds.
  • substantially as the term is used herein means completely or almost completely; for example, a composition that is "substantially free” of a component either has none of the component or contains such a trace amount that any relevant functional property of the composition is unaffected by the presence of the trace amount, or a compound is “substantially pure” is there are only negligible traces of impurities present.
  • an “effective amount” or a “therapeutically effective amount” of an agent refers to an amount of the agent that alleviates, in whole or in part, symptoms associated with the disorder or condition, or halts or slows further progression or worsening of those symptoms, or prevents or provides prophylaxis for the disorder or condition, e.g., an amount that is effective to prevent, inhibit or treat in the individual one or more symptoms.
  • a “therapeutically effective amount” refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired therapeutic result.
  • a therapeutically effective amount is also one in which any toxic or detrimental effects of the agent(s)are outweighed by the therapeutically beneficial effects.
  • sequence refers to a nucleotide sequence of any length, which can be DNA or RNA; can be linear, circular or branched and can be either single-stranded or double stranded.
  • donor sequence refers to a nucleotide sequence that is inserted into a genome.
  • a donor sequence can be of any length, for example between 2 and 10.000 nucleotides in length (or any integer value therebetween or thereabove), e.g., between about 100 and 1,000 nucleotides in length (or any integer therebetween), e.g., between about 200 and 500 nucleotides in length.
  • Binding refers to a sequence-specific, non-covalent interaction between macromolecules (e.g., between a protein and a nucleic acid). Not all components of a binding interaction need be sequence-specific (e.g., contacts with phosphate residues in a DNA backbone), as long as the interaction as a whole is sequence-specific. “Affinity” refers to the strength of binding: increased binding affinity being correlated with a lower Kd.
  • a “binding protein” is a protein that is able to bind non-covalently to another molecule.
  • a binding protein can bind to, for example, a DNA molecule (a DNA-binding protein), an RNA molecule (an RNA-binding protein) and/or a protein molecule (a proteinbinding protein).
  • a DNA-binding protein a DNA-binding protein
  • RNA-binding protein an RNA-binding protein
  • a proteinbinding protein In the case of a protein-binding protein, it can bind to itself (to form homodimers, homotrimers, etc.) and/or it can bind to one or more molecules of a different protein or proteins.
  • a binding protein can have more than one type of binding activity.
  • a “homologous, non-identical sequence” refers to a first sequence which shares a degree of sequence identity with a second sequence, but whose sequence is not identical to that of the second sequence.
  • a polynucleotide comprising the wild-type sequence of a mutant gene is homologous and non-identical to the sequence of the mutant gene.
  • the degree of homology between the two sequences is sufficient to allow homologous recombination therebetween, utilizing normal cellular mechanisms.
  • Two homologous non-identical sequences can be any length and their degree of non-homology can be as small as a single nucleotide (e.g., for correction of a genomic point mutation by targeted homologous recombination) or as large as 10 or more kilobases (e.g., for insertion of a gene at a predetermined ectopic site in a chromosome).
  • Two polynucleotides comprising the homologous non-identical sequences need not be the same length.
  • an exogenous polynucleotide i.e., donor polynucleotide
  • an exogenous polynucleotide i.e., donor polynucleotide of between 20 and 10,000 nucleotides or nucleotide pairs can be used.
  • a “target site” or “target sequence” is a nucleic acid sequence that defines a portion of a nucleic acid to which a binding molecule will bind, provided sufficient conditions for binding exist.
  • HLA human leukocyte antigen
  • genes/associated genes e.g., beta-2 microglobulin
  • other genes have been knocked in (e.g., PDL1, CTLA4-Ig, CD47, and HLA-E, for example).
  • HLA-E human leukocyte antigen
  • These approaches are effective at abrogating the adaptive (T and B cell mediated) and one innate cell type (NK-cell mediated) immune responses.
  • NK-cell mediated innate cell type
  • compensatory mechanisms by which the recipient’s immune cells could still reject the transplanted graft (indirect pathway of allorejection) and/or loss of protection against uncontrolled tumor growth and/or viral infection.
  • the present methods may be a “gentler” approach that maintains some degree of anti-tumor and/or anti-viral response, while also diminishing the allorejection response.
  • ICM-1 INF-inducible factor-1
  • E-selectin and P-selectin or other adhesion molecules as well as associated inflammatory molecules, e.g., interferon gamma, MCP-1 and other chemoattractants
  • pro- regulatory cytokines e.g., IL10
  • chemoattractants may be added to the cells (knockin).
  • CD54 an adhesion molecule, CD54 (or ICAM-1), was gene edited out of induced pluripotent stem cells (iPSCs), producing a knock-out line, using CRISPR/Cas9 editing approaches.
  • iPSCs induced pluripotent stem cells
  • CD54 is involved in the adherence of immune cells to parenchymal cells and to antigen presenting cells. It plays a key role in the formation of the immune synapse during cell attachment/killing of many cell types, as well as in the tethering, rolling, and extravasation process by which immune cells interact with endothelial cells. Also, there are associations between CD54 and inflammation, whereby knock-out of CD54 may positively- impact anti-inflammatory responses of the cells.
  • GSH glutathione
  • sgRNA identification for the site of interest was accomplished using the CRISPOR design tool described in the art.
  • the sgRNAs for use were a 1.5 nmol synthetic sgRNA with 2’-O-methyl 3’ phosphorothioate modification at the first and last 3 nucleotides.
  • Electroporation, selection, and growth iPSCs were cultured in mTeSRTMPLUS media (StcmCcll Technologies) on Matrigcl® until -80% conflucncy following standard cell culture protocols. 24 hours before electroporation, cells were treated with CloneRTM (StemCell Technologies) following manufacturer protocol.
  • the sgRNA constructs were reconstituted following manufacturer protocols to a concentration of 150 pmole/pL.
  • 1 pL of reconstituted sgRNA was pooled with 4 pg Cas9 Nuclease protein (TrueCutTM Cas9 Protein V2, Thermo Fisher Scientific) and 5 pL of NeonTM Buffer R (Invitrogen) to promote Cas9-RNP complex formation.
  • 1.0 pL of ssODN primer reconstituted to 1 pg/pL concentration, designed with homology overhangs of at least 40 base pairs was added to the Cas9-RNP mix.
  • Cells were singularized and lifted with a 1: 1 mixture of 0.5 mM EDTA: Accutase® for 3-4 minutes, resuspended in 1 mL PBS, and pelleted. Approximately 400,000 cells were resuspended in 35 pL NeonTM Buffer R and mixed with 8 pL of the pre -prepared Cas9-RNP complex with repair ssODN. Cells were electroporated with a 10 pL NEON electroporation format using 1200V, 30 msec, lx pulse settings.
  • Genotyping Bulk gDNA was collected from dissociated cells using QuickExtractTM DNA Extraction Solution 1.0 (Epicentre) to confirm editing efficiency prior to clonal selection. Single-cell clones were manually selected and mechanically disaggregated. Genomic DNA was isolated from a portion of these clones using QuickExtractTM DNA Extraction Solution 1.0 (Epicentre).
  • Genotyping primers were designed flanking the mutation site, allowing amplification of this region using Q5® polymerase-based PCR (NEB). PCR products were identified via agarose gel and purified using a ZymocleanTM Gel DNA Recovery Kit (Zymo Research). Clones were submitted to Quintara Biosciences for Sanger sequencing to identify clones with the proper genetic modification.
  • Offtarget analysis To identify whether the CRISPR-Cas9 system produced any nonspecific genome editing, suspected off-target sites for genome modification were analyzed. Using the 5 highest- likelihood offtarget sites for each sgRNA predicted by the CRISPOR algorithms, genotyping primers were prepared to amplify these regions via Q5®-polymerase PCR. PCR products were identified via agarose gel, purified using a ZymocleanTM Gel DNA Recovery Kit, and submitted to Quintara Bioscicnccs for Sanger sequencing.
  • PSC- CVTs PSC-based cardiovascular therapy
  • Different differentiated cells may be employed in that type of therapy.
  • the primary interface of transplant tolerance and rejection, endothelial cells are targeted by genetically interrupting the ability of allogeneic adaptive and innate immune cells to adhere to, infiltrate, and destroy the graft.
  • Hypoimmune grafts optionally having a combination of different cell types, each of which is obtained from one or more adhesion molecule knock out stem cells/stem cell lines are assessed for allo-immunogenicity, for example, in a humanized mouse model, e.g., the NeoThy model which is a more-robust (non-fetal) alternative to the gold-standard, but suboptimal, bone-marrow-liver-thymus (BLT) fetal model.
  • a humanized mouse model e.g., the NeoThy model which is a more-robust (non-fetal) alternative to the gold-standard, but suboptimal, bone-marrow-liver-thymus (BLT) fetal model.
  • adhesion molecules e.g., ICAM-1
  • AECs PSC-derived arterial endothelial cells
  • Figure 1 targeted disruption of leukocyte adhesion facilitates immune tolerance of vascularized tri-cellular PSC-CVTs made from gene-edited PSC-AECs, cardiomyocytes (CMs), and cardiac fibroblasts (C-fibs). Those grafts are tested for function and immunogenicity.
  • PSC-AD-CVT grafts that are immune-tolerated in vivo in the NeoThy humanized mouse model.
  • PSC-AD-CVT grafts composed of ratios (e.g., 1x105 in 1: 1: 1 ratios, then doubling each type one by one) of hypoimmune PSC-AECs, -CMs, and -C-Fibs form vascularized cardiac organoids with contractile function, that are immune- tolerated in the NeoThy model (e.g., diminished graft immune cell infiltration).
  • ratios e.g., 1x105 in 1: 1: 1 ratios, then doubling each type one by one
  • hypoimmune PSC-AECs, -CMs, and -C-Fibs form vascularized cardiac organoids with contractile function, that are immune- tolerated in the NeoThy model (e.g., diminished graft immune cell infiltration).
  • PSC Pluripotent stem cell
  • PSC-CVTs Pluripotent stem cell-based cardiovascular therapies
  • iPSCs induced PSCs
  • CMs peripheral blood T cells
  • ECs endothelial cells
  • HLA-banking faces a number of hurdles (e.g., ensuring representation for genetically-diverse racial and ethnic minorities).
  • Research has shifted to the development of universal hypoimmune PSCs, whereby one cell line could be used for most, if not all, patients in the country, including those with rare HLA types.
  • a hypoimmune PSC-CVT for MI for use in any form of heart disease where damaged and/or pathologic heart tissue needs to be replaced with vascularized, functional cells that are tolerated by the recipient’s immune system, is provided.
  • the approach is designed to be tolerated by both adaptive (e.g., T cells) and innate immune cells (e.g., monocytes), the latter of which are not directly targeted in current hypoimmune PSC therapies.
  • the graft provides for treatment for cardiac pathologies that kill and/or diminish the quality of life for millions of people worldwide.
  • First-generation (l st -gen) hypoimmune gene-edited cells e.g., knock-out [KO] of MHC class I+II
  • CMs CMs
  • HLA ablation and related published strategies are intended to diminish adaptive immunity (T cells, donor specific antibody) and NK cells, and they neglect other innate immune cells (e.g., monocytes) that play key roles in allorej ection.
  • One approach to gene-editing, as described herein, is to focus on targeted gene edits important for the cell biology and immune cell interactions of individual cell types (e.g., adhesion of adaptive and innate immune cells to arterial endothelial cells [AECs]), which are also important in effector immune synapse formation with other parenchymal cells (e.g., CMs).
  • AECs adaptive and innate immune cells to arterial endothelial cells
  • CMs parenchymal cells
  • ECs are at the primary interface of where transplant rejection begins, and/or immune tolerance is maintained.
  • the PSC-CVTs described herein include AECs, which form the luminal barrier in larger blood vessels of the heart (e.g., coronary arteries), and, critically, have a lower baseline propensity for leukocyte adhesion than vascular or other EC subtypes .
  • Targeted disruption of IC AM- 1 -mediated leukocyte: endothelial adhesion may confer immune tolerance to the AECs, and to CMs and cardiac fibroblasts (C-fibs) within the PSC-CVT graft, by preventing initial leukocyte attachment and immune synapse formation.
  • KO of donor adhesion molecules resulted in diminished allogeneic T cell responses, nearly double the survival time of allomismatched hearts compared with wild-type (WT) grafts, and, importantly, KO did not cause major pathogenic vascular defect and/or embryonic lethality . Additionally, since ECs serve as semi-professional antigen presenting cells, the approach of gene-editing to prevent stable leukocyte:EC interactions described herein has potential for disrupting effector memory T cell function.
  • Figures 1A and B show that gene expression of ICAM-1 increases during activation/inflammation, as well as after addition of inflammatory stimulus (e.g., TNFa), even in beta-2 microglobulin (B2M) KO l st -gen PSC-AECs lacking surface MHC class I, similar to published reports with immortalized mouse and primary human ECs. This further validates the strategy of focusing on molecules that a role in allorejection, and suggests that adhesion molecules (and the innate immune cells that bind to them) may still contribute to allorejection in B2M KO PSC grafts.
  • inflammatory stimulus e.g., TNFa
  • the KO approach will likely confer further adaptive and innate immune protection to PSC-CMs, AECs, and -C-fibs within the grafts.
  • grafts are prone to arrhythmias post-transplantation, a dangerous adverse event thought to be associated with the immature developmental status of the PSC-CMs used, cellular impurities, and/or the absence of homeostatic cues from accessary cell types (e.g., C-Fibs) present in normal heart but lacking in relatively homogeneous populations of PSC-CMs.
  • the PSC-CVT graft design disclosed herein uses an intentional mixture of highly-pure (to avoid teratomas) CMs and C-fibs (to provide homeostatic cues and aid in maturation), utilizing advanced protocols .
  • a third, specialized purified cell population, AECs is then added to promote neovascularization, CM maturation, and homeostatic cross-talk.
  • the tri-cellular hypoimmune grafts are relatively more mature, and have high potential for clinical translation with long-term function and immune tolerance. Input ratios of these individual cell types (CMs, C-Fibs, AECs) can be varied in in vivo studies.
  • Figures 2A and B show robust leukocyte adhesion gene-edited PSC-AD-CVT grafts that demonstrate identity, function (e.g., contractility) and neovascularization upon transplantation into humanized mice.
  • MLR data in Figure 3 show that l st -gen B2M KO PSC AECs are capable of eliciting a CD8+ T cell proliferative response in the presence of antigen presenting cells, indicating that while B2M KO may protect against direct pathway allorejection, as shown by significantly diminished proliferation in B2M KO versus WT cells, such l st -gen gene-edited PSC therapies may still be prone to the indirect pathway mediated chronic rejection seen in solid organ transplant patients. That is, there is still a large population of proliferating T CD8 + T cells versus unstimulated controls.
  • NeoThy A humanized mouse model, the NeoThy, was employed. Humanized mice are a powerful research tool for modeling the in vivo human immune response to PSC transplants.
  • the existing gold-standard humanized mouse, the fetal-tissue based “BLT” has been reported to be suboptimal for allorejection studies due to naive and regulatory T bias.
  • the NeoThy in contrast, has a naive CD4 + T cell compartment more similar to adult patients than does the BLT model ( Figure 4 A), and is a high-fidelity model of allorejection (e.g., extensive immune infiltration) of transplanted PSC-CM grafts ( Figure 4B).
  • a hypoimmune PSC-AD-CVT graft with tri-cellular composition, having a lack of alloreactivity in vitro and in vivo, may be employed to restore function post-MI, in the absence of in vivo alloreactivity, e.g., as tested in an advanced NeoThy humanized mouse MI model which is a testing platform for hypoimmune PSC therapies, which may be useful for many patients with MI and other pathologies characterized by cellular dysfunction in immune-competent anatomical sites.
  • MI is a devasting pathology, resulting in death or diminished quality of life for millions of patients.
  • Existing therapies are suboptimal, whereas PSC-based cellular therapies have great potential for improving and saving lives, especially if they are durably immune- tolcratcd.
  • Scalability and pluripotent differentiation potential arc two key advantages of PSCs over other primary cell-based CVTs.
  • the status quo as it pertains to gene-edited PSC therapies is use of HLA I+II KO lines, a promising but generalized approach that does not take into account nuanced cell-type specific interactions with the immune system and leaves grafts vulnerable to rejection mediated by the indirect pathway, in concert with monocytes and other innate immune cells.
  • a gene-editing strategy is employed focused on preventing immune cell adhesion, at the endothelial cell: immune interface and in adaptive and innate immune synapses important for parenchymal cell rejection/tolerance.
  • a tri-cellular PSC-AD-CVT graft for superior reparative function and hypoimmunogenicity is prepared.
  • the human immune response is interrogated with a multi-faceted approach that utilizes classic transplant immunology techniques and stem cell biology, and leverages an advanced in vivo humanized mouse model. The data allow for a better understanding of the mechanisms of leukocyte adherence to AECs, and those mediating activation and inflammation during the allorejection process.
  • the NeoThy model provides the regenerative medicine community with a tool for long-term cardiomyopathy studies in the context of the human immune system, which is not feasible with other (e.g., BLT-type) humanized mice.
  • PSC differentiation protocols create tri-cellular PSC-AD-CVT grafts, which, combined with robust evaluation of in vitro and in vivo human immunogenicity, can further clinical use of reparative and durably tolerated PSC-CVTs.
  • ICAM-1 KO PSC- AECs, CMs, and C-fibs have normal phenotypic identity.
  • PSC-AD-CVTs are immune-tolerated compared to WT. • Addition of ICAM-1 KO to first-generation hypoimmune PSC-CVTs improves immune- tolerance.
  • the hypoimmune. tri-cellular PSC-AD-CVT incorporates genetic ablation of a crucial cell adhesion molecule, ICAM-1, that is used by multiple adaptive and innate immune cells during key steps of allorejection.
  • ICAM-1 crucial cell adhesion molecule
  • the therapy design and immunogenicity assays address and experimentally interrogate the innate, as well as adaptive, immune responses.
  • the immunomodulatory gene edits do not significantly disrupt the phenotype and function (e.g., diminish the reparative capacity) of the cells.
  • the gene- edited PSCs After preparing the gene- edited PSCs, they are differentiated into the three PSC-CVT cell types, then phenotypic, functional, and immunogenicity studies are conducted for the individual gene-edited cell types to characterize adhesion molecules in interactions between immune cells and AECs, CMs, and C- fibs, and determine how ablation of ICAM-1 impacts independent cell biological function. This allows for durably-tolerated PSC-CVTs that can improve the health and well-being of millions of MI patients worldwide.
  • ICAM-1 deletion does not meaningfully alter phenotype or impair function of PSC- AECs, -CMs, or -C-fibs. Additionally, immune cell interactions with each of the three cell types are diminished and there is concordant diminishment of in vitro measures of allorejection. These effects are apparent with an ICAM-1 KO alone, and enhanced when combined with l st -gen gene- edited lines (i.e., ablation of MHC class I + II, addition of HLA-Edimer).
  • ICAM-1 KO PSC-AECs, CMs, and C-fibs have normal phenotypic identity: CRISPR/Cas9-based gene editing is efficient.
  • CRISPR/Cas9-based gene editing is efficient.
  • a high-fidelity Cas9 variant is used for knockout of ICAM-1 plus introduction of a constitutively- expressed Akaluc luminescence reporter for use in downstream bioluminescent imaging (BLI) studies.
  • the H9 B2M KO/CIITA KO/HLA-E dimer (H9 KO Edimer) line may be used as a l st -gen control line.
  • ICAM-1 KO lines are edited, and verified as selected for KO via IFNy based upregulation of ICAM-1 in untargeted cells. Multiple KO clones are banked and karyotyped and screened for off-target activity. Four iterations of PSCs are used for downstream experiments: 1) ICAM-1 KO alone, 2) l st -gcn KO Edimcr alone, 3) ICAM-1 KO added to l st -gen KO Edimer, and 4) WT (unedited) H9. Studies on multiple l st -gen hypoimmune PSC lines showed that they are capable of differentiation into phenotypically normal and functional AECs and CMs (Figure 5).
  • PSC lines or H9 with KO of other adhesion molecules may also be prepared, e.g., double KO of E-Selectin and P-selectin.
  • PSC-AD-CVTs have normal cell-type specific function: In addition to cell phenotype and morphology, normal cell-type specific function post-KO is shown. Methods previously described validate gene-edited PSC-AECs, PSC-CMs, and PSC-C-fibs. Briefly, normal AEC function are verified by assessing oxygen consumption rates, nitric oxide production levels, and shear stress responses; CMs by macroscopic contractility; and C-fibs by immunolabeling to demonstrate extracellular matrix formation via staining of collagen I and fibronectin.
  • PSC-AD-CVTs are immune-tolerated compared to WT: In vitro assessment of immunogenicity is one step in the evaluation of the tolerogenic potential of the disclosed PSC- AD-CVTs.
  • Adaptive e.g., T cells
  • innate immune cells e.g., monocytes
  • ICAM-1 e.g., ICAM-1
  • 6-day MLRs are conducted based on a previously published protocolwith allogeneic peripheral blood mononuclear cells (PBMCs) (containing the above three cell populations) and each individual gene-edited PSC-derived cell above as targets.
  • PBMCs peripheral blood mononuclear cells
  • proliferation is measured by flow cytometry on gated cell subtypes (e.g., effector memory T cells), and LAMP1 and T cell/NK cytotoxic activity is assessed in flow cytometry and CytoTox 96® assays, respectively.
  • PD1, LAG3, and other markers of T cell exhaustion are measured to evaluate the effect of ICAM-1 ablation on effector function.
  • MCP-1 monocyte chemoattractant protein 1
  • Figure 3 shows that l st -gen (B2M KO) hypoimmune PSC-AECs are more- weakly immunogenic than WT cells, but that there is still a proliferative response in MLRs, indicative that the indirect pathway of allorejection may be involved.
  • An existing l st -gen KO Edimer PSC line is edited to also incorporate ICAM-1 KO, with the intention of further diminishing the rejection response by preventing effective adhesion.
  • T cells With ICAM-1 KO cells, a decreased adhesion of T cells, B cells, NK cells and monocytes is observed in immunofluorescence microscopy and flow cytometry-based studies, as well as a decreased immune response in MLR assays. An increased frequency of T cells with exhausted phenotype mediates diminished direct and indirect pathway responses.
  • PSC-AD-CVT grafts composed of ratios of hypoimmune PSC-AECs, -CMs, and -C-Fibs form vascularized cardiac organoids with contractile function in the heterotopic kidney capsule site of immune-deficient mice.
  • PSC-AD-CVT grafts are immune-tolerated in the minimally -inflamed environment of the NeoThy model, in the short and long-term.
  • NeoThy overcomes a weakness of other humanized mice — graft vs host disease (GVHD). GVHD creates systemic inflammation that interferes with the discernment of experimental design vs model-inherent immune effects, and shortens experimental windows by causing premature mouse deaths.
  • GVHD graft vs host disease
  • NBSGW mouse which does not require irradiation for humanization, when used for creation of the NcoThy, in addition to anti-CD2 passenger thymocyte depletion, has a significant reduction in premature death and a concordantly large experimental window for long-term transplantation studies, demonstrated in Figure 7.
  • the use of the NBSGW mouse allows for control the inflammatory milieu and to conduct long-term studies (there are few published PSC transplantation studies with >30 day timepoints, diminishing their relevance for chronic rejection assessment).
  • the input cell ratios of the tri-cellular PSC-AD-CVT are varied in non-humanized NBSGW mice to create contracting vascularized grafts shown in Figure 2.
  • the immunogenicity of the graft is assessed in the NeoThy model.
  • PSC-AD-CVTs are transplanted in the heterotypic kidney capsule site ( Figure 2) which is a blood rich location, to investigate graft function as well as the interactions between graft and human immune cells. Graft composition and immunogenicity are interrelated. Hypoimmune grafts are functional in non-humanized and NeoThy mice, and in NeoThy mice, the grafts are tolerated by an allogeneic human immune system.
  • PSC-AD-CVT grafts composed of ratios of hypoimmune PSC-AECs, -CMs, and -C- Fibs form vascularized cardiac organoids with contractile function in the heterotopic kidney capsule site of immune-deficient mice.
  • the human heart is composed of multiple cell types, present in various frequencies, which work synergistically to maintain homeostasis and proper biological function within the cells themselves and the organ as a whole.
  • the tri-cellular PSC-AD-CVT utilizes highly-pure PSC-derived AECs, CMs, and C-fibs so that the proportion of these cells (and minimize undefined populations with unknown function and/or teratoma potential) are controlled, in order to achieve engraftment and reparative function.
  • the input ratios of the three graft cell types are varied, then the grafts are transplanted in a spheroid format (Figure 3) shown to produce engraftment in the kidney capsule (heterotopic) and heart (orthotopic) of immune-deficient mouse hosts, respectively.
  • Figure 3 A matrix that varies cell number and spheroid size, prior to transplantation, is used.
  • the preparations of PSC-AD-CVT spheroids are assessed for macroscopic contractility before being transplanted under the kidney capsule of 8-week-old NBSGW immune-deficient mice. Graft integrity/engraftment is monitored by weekly BLI tracking, via the Akaluc reporter co-engineered into the hypoimmune PSCs ( Figure 8).
  • mice that are graft positive by BLI are anesthetized at 1 month, and undergo survival surgery to observe graft size, vascularization, and contractility rate. At 3 months, the procedure is repeated and animals arc then sacrificed/tissue collected after recording observations. Tissues are assessed by histology (for immune infiltrate and evidence of teratoma) and RNA sequencing for CM, AEC, and C-fib markers (e.g., cardiac troponin T [cTNT], CD31) and maturation- associated genes in comparison with dO grafts retained as controls. A small graft sample for all transplant experiments is retained for other studies analyzing propensity for viral infection.
  • CM for immune infiltrate and evidence of teratoma
  • C-fib markers e.g., cardiac troponin T [cTNT], CD31
  • PSC-AD-CVTs are compared with WT tri-cellular PSC-CVTs and PSC-CMs as controls. Robust engraftment of vascularized, contracting grafts in PSC-AD-CVTs and WT PSC-CVTs is observed, and both tri- cellular grafts are larger than PSC-CM alone grafts.
  • the grafts mature in vivo when compared to dO to 3-month timepoint gene expression signatures of genes associated with cardiac maturation (e.g., SCN5A, GJA1, KCNJ4), similar to in vitro studies showing time-associated maturation changes (Figure 9).
  • NeoThy humanized mice humanized with an allogeneic immune system (complete HLA mismatch).
  • a graft candidate(s) determined by graft size, vascularization, contractility, and maturity
  • NeoThy humanized mice humanized with an allogeneic immune system (complete HLA mismatch).
  • an advantage of the NeoThy is the ability to conduct long-term studies (e.g., over 3 months) vs. traditional fetal tissue-based models which can often result in loss of animals and experimental power due to premature GVHD-associated mouse death.
  • the NeoThy model was used to assess a short-term time point (1 month) commonly used in the humanized mouse and/or gene-edited PSC literature, and also a long-term time point (3 months) that allows for determining susceptibility to chronic allorej ection.
  • Systemic inflammation was assessed by Luminex ( Figure 6) pre- and post-transplant.
  • Effector memory T cells were quantified by flow cytometry, including being analyzed for markers of T cell activation and exhaustion.
  • Transplanted PSC-AD-CVTs were assessed for infiltration of human CD4+, CD8+, and FoxP3+ T cells as well as for human monocytes/macrophages. Tissue fibrosis and anatomical integrity were assessed by histopathology.
  • PSC-AD-CVT was tested for reparative function, and immune tolerance potential, in a high-fidelity model that mimics the inflamed and complex physiological environment seen of an MI patient’s heart. That is, the hypoimmune PSC-AD-CVT was tested whether it is immune- tolerated in a robust, clinically-relevant in vivo environment. Demonstration of reparative function that persists upon encounter with the complex post-MI human immune response is a clear indication of translational potential. While immune-deficient animals are receptive to transplanted xenogeneic tissues, their response to injury differs from immune-competent WT animals. The present model reconstitutes a functional human immune system, more closely modeling the normal human immune roles in cardiac injury and transplantation. The immune- tolerated PSC-AD-CVT has use in a multitude of patient populations, requiring little to no immunosuppressive medication.
  • NeoThy MI model was employed, whereby MI is induced in human-chimeric animals (16 weeks post-humanization) by left anterior descending (LAD) artery ligation with a nonabsorbable suture, using lACUC-approved surgical techniques and pain-relief measures. Baseline inflammation was assessed by Luminex® assay of peripheral blood, in comparison to non-MI (sham surgery, without ligation step) NeoThy mice of comparable age and time post-humanization, as well as naive NBSGW controls.
  • Echocardiographic imaging was performed, and images were analyzed for MI via calculation of the left ventricular ejection fraction, fractional shortening, end-diastolic volume, and end-systolic volume. Additionally, after 4 weeks, immuno staining of murine hearts was conducted to assess infarct size, decreased ventricular wall thickness, and apoptosis (TUNEL assay). MI resulted in increased human inflammatory cytokines (e.g., IFNy, ILip, TNFoc) and infiltration of human CD1 lb + CD33 + CD16 + macrophages. Activated effector memory CD4 + and CD8 + T cells in the periphery and inflammation-induced clonal expansion were detectable by Adaptive Biotechnologies TCRP chain rearrangement sequencing, as shown in Figure 10.
  • PSC-AD-CVTs Post-MI, orthotopic transplant of gene-edited PSC-AD-CVTs results in vascularized grafts that improve cardiac function, decrease infarct size, and increase ventricular wall thickness: PSC-AD-CVTs were transplanted into MI non-humanized NBSGW and humanized NeoThy mice, with Mis introduced using the same methodology described above. Five to ten spheroids were suspended in a fibrin matrix patch, positioned over the site of infarction, immediately following LAD ligation. Animals were monitored for engraftment by weekly BLI monitoring.
  • Gene-edited PSC-AD-CVTs are durably tolerated by allogeneic human adaptive and innate immune cells in the inflammatory post-MI humanized mouse heart: In addition to the engraftment and reparative function in non-humanized NBSGW mice, the hypoimmune PSC- AD-CVTs also yield the same engraftment and reparative results in NeoThy mice, whereas WT PSC-CVTs and l st -gen gene-edited PSC-CVTs, to a lesser degree, are rejected by the allogenic human immune system.
  • Pluripotent stem cell (PSC)-derived cell therapies are promising reparative treatments for a variety of cardiovascular diseases that kill over 655,000 Americans each year, and preventing their immune rejection post-transplant is crucial for effective clinical translation. Due to their scalability, which enables large-scale cell banking, PSCs are an ideal cell source for gene-editing approaches to improve transplantation outcomes and achieve immune tolerance. Recently, multiple research groups have created gene-edited hypoimmune PSCs (e.g., knockout [KO] of human leukocyte antigen [HLA]) capable of evading allorejection by T cells, donor specific antibodies, and/or natural killer cell-mediated cytotoxicity in short-term studies.
  • KO knockout
  • HLA human leukocyte antigen
  • PSC-based cell therapies that are immune-tolerated, have little-to-no sustaining need for immunosuppression, and that meaningfully improve patient health and quality of life are described herein.
  • CRISPR/Cas9 gene-editing approaches are used to target adhesion molecules (AMs) (e.g. ICAM-1) on human PSC-derived cardiovascular- therapies (CVTs) to disrupt the adherence, infiltration, and destruction of vascularized grafts by allogeneic immune cells; and 2) cellular composition and immunogenicity profiles of next-generation hypoimmune PSC-CVT grafts are investigated for their reparative capacity in the inflammatory setting of myocardial infarction (MI).
  • MI myocardial infarction
  • AM genes facilitate immune tolerance of PSC-CVTs via two mechanisms: 1) diminished immune cell contact-mediated destruction; and 2) anti-inflammatory effects (e.g., secreted factor and gene expression changes) directly associated with genetically disrupting AM function.
  • Hypoimmune PSCs are a clinical platform , e.g., for immune tolerance of PSC grafts.
  • inflammatory responses initiated by immune cell:PSC-CVT graft interactions Preliminary data demonstrated a direct relationship between inflammatory stimulation (TNFa) and ICAM-1 function in PSCs, and indicate that ICAM-1 deletion increases PSC free radical scavenging potential.
  • Adaptive and innate immune cells may release lesser amounts of pro-inflammatory cytokines and chcmokincs and more anti-inflammatory factors upon in vitro interaction with ICAM-1 KO cells.
  • KO cells inherently may be more resistant to inflammatory cues (e.g., oxidative stress) vs. WT cells.
  • MI may induce systemic inflammatory cytokines in the humanized NeoThy mouse model.
  • a gene-edited PSC-CVT may show clear evidence of immune-tolerance.
  • the results contribute data on the mechanisms of PSC immunogenicity and transplant tolerance, and validating the gene-editing approach of directly targeting graft:immune cell adhesion and associated inflammatory pathways.
  • PSC Pluripotent stem cell
  • PSC-CVTs Pluripotent stem cell-based cardiovascular therapies
  • iPSCs induced PSCs
  • HLA human leukocyte antigen
  • a gene-edited PSC-CVT for MI with potential for future use in any form of heart disease where damaged and/or pathologic heart tissue needs to be replaced with vascularized, functional cells that are tolerated by the recipient’s immune system.
  • the approach targets adhesion molecules (AMs), specifically ICAM-1, which is a key AM involved in immune cell binding and/or inflammation.
  • the approach is designed for tolerance by both adaptive (e.g., T cells) and innate immune cells (e.g., monocytes), the latter of which are not directly targeted in currently published hypoimmune PSC therapies (e.g., HLA knockout [KO]).
  • a gene-edited PSC-CVT is useful for the treatment for cardiac pathologies that kill and/or diminish the quality of life for millions of people worldwide, in addition to validating an entirely new targeting approach for hypoimmune gene-editing.
  • next-Generation Gene-Edited Hypoimmune PSC-CVTs Recently, first-generation (Ist-gen) gene-edited hypoimmune cells (e.g., KO of HLA class I+II) were described that are able to evade recognition by T cells, donor specific antibodies, and/or natural killer (NK) cell- mediated cytotoxicity in short-term studies. These gene-edited cells have been used for differentiation into multiple PSC-derived cell types, including CMs. These cells maintain their phenotype and functional attributes, and in purified formats do not develop teratomas following gene-editing.
  • Ist-gen first-generation gene-edited hypoimmune cells
  • CMs natural killer
  • grafts are prone to arrhythmias post-transplantation, a dangerous adverse event thought to be associated with the immature developmental status of the PSC-CMs used, cellular impurities, and/or the absence of homeostatic cues from accessory cell types (e.g., C-Fibs) present in normal heart but lacking in relatively homogeneous populations of PSC-CMs.
  • accessory cell types e.g., C-Fibs
  • the present PSC-CVT graft design uses an intentional mixture of highly-pure (to avoid teratomas) CMs and C-fibs (to provide homeostatic cues and aid in maturation), utilizing advanced protocols.
  • AECs a third, specialized purified cell population, AECs, were added to promote neovascularization, CM maturation, and homeostatic cross-talk.
  • the hypoimmune tri-cellular grafts described herein may be more mature than CMs alone, and have high potential in the clinic because of long-term functional and immune tolerance capacities.
  • Targeting Immune Cell Adhesion in Allorejection A strategy for achieving PSC- CVT tolerance couples cutting-edge CRISPR/Cas9 gene-editing techniques with the lessons of over 50 years of transplantation immunology. Importantly, there is a focus specifically on the unique biology of AMs as an alternative target vs. global HLA ablation, the latter of which may be too extreme (e.g., could render the cells susceptible to malignancy). In one aspect, the disclosure provides for attenuating, but intentionally not completely ablating, immune cell adhesion and pathological inflammatory responses.
  • the tri-ccllular PSC-CVT grafts include AECs, which form the luminal barrier in larger blood vessels of the heart (e.g., coronary arteries), encouraging post-transplant neovascularization.
  • AECs form the luminal barrier in larger blood vessels of the heart (e.g., coronary arteries), encouraging post-transplant neovascularization.
  • the vasculature is the primary interface of transplant rejection and, critically, AECs have a lower baseline propensity for leukocyte adhesion than vascular or other EC subtypes.
  • ICAM-1 is involved in post-transplantation immune interactions with the vasculature (tethering, rolling, extravasation) as well as cytolysis during immune cell killing (immune synapse formation).
  • LFA-1 is the primary ligand for ICAM-1 and it is expressed on adaptive (e.g., T cells) and innate (e.g., monocytes and dendritic cells) immune cells, all of which play critical and coordinated roles in allorejection.
  • Figure 16 shows the mixed lymphocyte reaction (MLR) assay that ca be used to experimentally test immunogenicity.
  • MLR mixed lymphocyte reaction
  • Controlled and highly-pure WT and KO target cell populations allow for successful clinical translation of PSC-CVTs, and also provides insights into traditional solid organ allorejection mechanisms.
  • High fidelity in vitro assays and in vivo models relevant to human immunology, including a humanized mouse model, are employed.
  • Immune cell-derived inflammatory cytokines and/or oxidative stress can initiate inflammatory responses in endothelial and other graft cells, which in turn feedback (in association with AMs) via additional release of cytokines and radical oxygen species that can further degrade graft cell viability.
  • endothelial and other graft cells which in turn feedback (in association with AMs) via additional release of cytokines and radical oxygen species that can further degrade graft cell viability.
  • AMs play important roles in responding to oxidative stress during inflammatory responses.
  • GSH glutathione
  • NeoThy Humanized mice are a powerful research tool for modeling the in vivo human immune response to PSC transplants.
  • NeoThy is in contrast to the existing gold-standard humanized mouse, the fetal-tissue based “BLT,” that has been reported to be suboptimal for allorejection studies due to naive and regulatory T cell bias.
  • BLT fetal-tissue based
  • the NeoThy has a naive CD4 + T cell compartment more similar to adult patients than does the BLT model, and is a high-fidelity model of allorejection (e.g., extensive immune infiltration) of transplanted PSC- CM grafts.
  • the NeoThy model may be a more replicable and reproducible system for broader uses and that such technologies would benefit from an investment to accelerate research in these areas.
  • the result is a hypoimmune PSC-CVT graft with a tri-cellular composition, having a lack of alloreactivity in vitro and in vivo. This hypoimmune graft strategy is pertinent for MI patients.
  • MI is a devastating pathology, resulting in death or diminished quality of life for millions of patients.
  • Existing therapies are suboptimal, whereas PSC-based cellular therapies have great potential for improving and saving lives, especially if they are durably immune - tolerated.
  • Scalability and pluripotent differentiation potential are two advantages of PSCs over other primary cell-based CVTs.
  • the status quo as it pertains to gene-edited PSC therapies is use of HLA T+II KO lines, a promising but biologically invasive approach that does not take into account nuanccd cell-type specific interactions with the immune system and leaves grafts vulnerable to rejection mediated by the indirect pathway, in concert with monocytes and other innate immune cells.
  • the grafts use a ngene-editing strategy focused on preventing allorejection mediated by immune cells, the disclosed tricellular graft allows for superior reparative function and hypoimmunogenicity; and the human immune response can be interrogated with a multifaceted approach that utilizes classic transplant immunology techniques and stem cell biology, and that also leverages an advanced in vivo humanized mouse model, thereby allowing for understanding the mechanisms of leukocyte adherence to vasculature and parenchymal cells, and mediating activation and inflammation during the allorejection process.
  • ICAM-1 KO will prevent allogeneic destruction of engraftable tri-cellular PSC-CVTs.
  • the hypoimmune, tri-cellular PSC-CVT incorporates genetic ablation of a crucial cell AM used by multiple adaptive and innate immune cells during key steps of allorejection.
  • ICAM-1 KO PSCs were generated with normal karyotype and typical differentiation yields of phenotypically normal PSC-CVT cell types.
  • Tri-cellular spheroids were generated that demonstrate contractility in vitro (Figure 13 A). Multiple gene-edited PSC lines were generated then differentiated into three PSC-CVT cell types, then assessed for immunogenicity of the individual gene-edited cell types in multiple assays. The mechanistic roles played by AMs in interactions between immune cells and AECs, CMs, and C-fibs, and how ablation of ICAM-1 impacts immune cell adhesion and downstream immunogenic responses, is defined in order to develop durably-tolerated PSC- CVTs that improve the health and well-being of millions of patients worldwide. KO of ICAM-1 results in diminished immune cell interactions and cell loss with each of the three cell types. Those effects with ICAM- 1 KO alone may be enhanced when combined with 1 st-gen gene- edited lines (i.e., ablation of MHC class I + II, addition of HLA-Edimer).
  • ICAM-1 KO lines were generated from multiple existing PSC lines, then KO verified via IFNy- and/or TNFa-based upregulation of ICAM-1 (see Figure 15). Multiple KO clones were banked, karyotyped and screened for off-target activity. Multiple l st -gen hypoimmune PSC lines were capable of differentiation into phenotypically normal and functional AECs and CMs ( Figure 15 and additional data not shown). Upon verifying normal karyotype by g-banding, our all PSC iterations are differentiated into AECs, CMs, and C-fibs and their phenotypes assessed.
  • RNAseq were used to verify that KO of ICAM-1, E- and P- selectin, and/or lst-gen gene targets do not result in off-target gene and protein expression effects or otherwise negatively impact the cells.
  • Normal cell-type specific function post-KO was demonstrated and gene-edited PSC-AECs validated, PSC-CMs, , and PSC-C-fibs. Briefly, normal AEC function was verified by assessing oxygen consumption rates; CMs by contractility; and C-fibs by immunolabeling to demonstrate extracellular matrix formation via staining of collagen I and fibronectin.
  • ICAM-1 KO PSC-CVT cell types elicit diminished alloimmune cell binding and proliferation compared to WT: In vitro assessment of immunogenicity is the first step in the methodical evaluation of the tolerogenic potential of the PSC-CVTs.
  • ICAM-1 plays a key role in adaptive (e.g., T cells) and innate immune cell (e.g., monocytes)) adherence to cellular targets via LFA-1 binding; therefore, in these cells as well as innate NK cells (mediators of missing-self cytotoxicity in solid organ transplantation and in lst-gen hypoimmune PSC therapies) were evaluated.
  • adaptive e.g., T cells
  • innate immune cell e.g., monocytes
  • PBMCs peripheral blood mononuclear cells
  • Figure 18 Data shows diminished binding of peripheral blood mononuclear cells (PBMCs) to ICAM-1 KO PSCs vs. WT ( Figure 18). It was demonstrated that AECs, CMs, and C-fibs differentiated from KO PSCs will also show attenuated immune binding and less target loss. 6- day MLRs, as shown in Figure 3, are conducted based on a previously published protocol with allogeneic PBMCs (containing the above three cell populations) and each individual gene-edited PSC-derived cell above as targets.
  • kidney capsule site is an accepting niche that is an anatomical site for assessing baseline engraftment and development in the absence of immune cells. Positive engraftment in the kidney indicates a likelihood of positive engraftment in the heart. Grafts were monitored via live-animal imaging BLI tracking of grafts ( Figure 19), as well as histologically 30 days posttransplant at experimental termination (described in more detail below).
  • GVHD graft vs host disease
  • GVHD creates systemic inflammation that interferes with the discernment of experimental design vs. model-inherent immune effects, and shortens experimental windows by causing premature mouse death.
  • Mouse host irradiation as well as anti-host passenger thymocytes within transplanted human thymus fragments are mediators of GVHD in humanized mice.
  • the NBSGW mouse does not require irradiation for humanization.
  • the NBSGW has significantly reduced premature death and a concordantly large experimental window for long-term transplantation studies.
  • CMs, AECs, c-Fibs mice per PSC cell type
  • NeoThy thus has utility for assessment of allorejection and tolerance of PSC-C VT cells transplanted hctcrotopically and provides data on the hypoimmunc status, and mechanistic immunogenicity studies of the complete PSC-C VTs transplanted orthotopically in the post-MI.
  • [00252] Define the inflammatory responses initiated by immune cell:PSC-CVT graft interactions in vitro, and in the post-MI NeoThy mouse in vivo'. Adaptive and innate immune cells release lesser amounts of pro-inflammatory cytokines and chemokines and more antiinflammatory factors upon in vitro interaction with IC AM- 1 KO cells, KO cells have more resistance to inflammatory cues vs. WT cells and MI induces systemic inflammatory cytokines in the humanized NeoThy mouse model.
  • Pathological inflammation plays a driving role in allorejection.
  • TNFa- induced ICAM-1 upregulation in Figure 1 AM cell biology and inflammatory stimulation are intimately connected. The direct cell binding-associated mechanisms of rejection is interrogated in order to focus on the cell adhesion component of the immune response (e.g., can we prevent lysis by ablating a key AM needed for T cell immune synapse formation?).
  • Inflammatory cytokines were measured in order to determine the role that ICAM-1 KO plays in the response to these cues, as the literature has shown an inverse relationship between ICAM-1 and the antiinflammatory response to oxidative stress in multiple cells types. KO of ICAM-1 conferred antiinflammatory benefits to PSC-CVTs that are inherent to the cells of the graft and also impact effector immune responses.
  • Adaptive and innate immune cell subtypes have diminished inflammatory cytokine responses to KO PSC-CVT cells and KO PSC-CVT cells have diminished inflammatory cytokine (protein and gene expression) responses following immune cell encounter: 18 hours MLR co-cultures of total PBMCs with ICAM-1 KO, Ist-gen KO, ICAM-1 KO + 1st gen KO- derived cells, ICAM-1 KO + E-/P-selectin, and WT PSC-CVT cell types were prepared. These short-term MLRs allow for the preservation of intact target cells, which is not possible in 6 day MLRs due to advanced target cell destruction.
  • Target cells were collected by enzymatic dissociation, and effector immune cells were removed via CD45 magnetic beads and stained for intracellular cytokine production (e.g., IFNy) by flow cytometry.
  • cytokine production e.g., IFNy
  • PSC-CVT cell types which also produce cytokines in response to inflammatory stress
  • RNAseq RNAseq vs. control cells without immune cell exposure. Identifying expression patterns of inflammation- resistant cells enables analysis of subpopulations via single cell RNAseq, which allows for evaluation of immune evasion mechanisms and design of hypoimmunc therapies.
  • FIG. 6 shows an example of one cytokine, monocyte chemoattractant protein 1 (MCP-1) that is produced in large quantities in 18-hour MLR co-cultures of WT PSC-AECs and allogeneic PBMCs.
  • MCP-1 monocyte chemoattractant protein 1
  • This finding illustrates the role of innate cells/monocytes in the natural alloresponse to unedited WT PSC-derived cells, which is diminished as a direct result of our AM gene-editing.
  • the analysis of immune cell intracellular cytokine flow and gene expression of target KO PSC-CVT therapies allows for the discemation of sources of supernatant cytokines.
  • T cells and monocytes secrete antiinflammatory cytokines (e.g., IL10), while the WT elicit relatively increased levels of inflammatory cytokines (e.g., IFNy, IL 1 [3, TNFa).
  • cytokines e.g., IFNy, IL 1 [3, TNFa.
  • specific cell types e.g., upregulated in CMs vs. AECs
  • sub-cell types via bulk and unique to the type of gene-edit (e.g., highest increase of MCP-1 in monocyte-alone co-cultures vs. T cell-alone for ICAM-1 KO cells, and the reverse for 1 st gen HLA KOs).
  • KO cells inherently will be more resistant to inflammatory cytokines vs. WT cells:
  • purified PSC-CVT cells alone were cultured in the presence of a panel of 20 individual cytokines, chemokines, and factors associated with the inflammatory immune responses of multiple immune cell types (e.g., IFNy, ILip, TNFa, IL17. TGF . IL15, MIP-la, RANTES).
  • RNAseq RNA collected for bulk RNAseq to analyze differences in cytokine/chemokine gene expression, inflammatory gene pathways, cell identity- associated genes to monitor for potential de-differentiation, and apoptosis/other mechanisms of cell death (e.g., NF-KB).
  • KO cells are more resistant to oxidative stress vs.
  • GSH levels were determined using ThiolTrackerTM dye (as shown in PSCs in Figure 17). Similar- elevated levels in the KO PSC-derived cells (e.g., CMs) were observed as was observed in the undifferentiated PSCs. Compounds known to cause oxidative stress, such as hydrogen peroxide and nitric oxide and damage associated molecular patterns (DAMPs), may be employed to interrogate whether the KO affords the cells better survival ability. Additionally, apoptosis of the cells is assessed via Caspase 3/7 and Annexin V/7-AAD. Diminished apoptosis in response to oxidative stress in the KO cells is observed relative to baseline in WT cells.
  • DAMPs damage associated molecular patterns
  • MI was induced in chimeric NeoThy animals (16 weeks post-humanization) by left anterior descending (LAD) artery ligation with a nonabsorbable suture, using lACUC-approved surgical techniques and pain-relief measures.
  • LAD left anterior descending
  • Systemic inflammation was assessed by Luminex® assay of peripheral blood pre-MI and post-MI in the same animals. All mice were of comparable human chimerism, age, and time post-humanization.
  • Echocardiographic imaging was performed, and images were analyzed for MI via calculation of the left ventricular ejection fraction, fractional shortening, end-diastolic volume, and end-systolic volume. Additionally, at the terminal time point of 30 days, in addition to collecting serum for inflammatory factor analysis, immunostaining of murine hearts was conducted to assess infarct size, decreased ventricular wall thickness, and apoptosis (TUNEL assay). MI resulted in increased human inflammatory cytokines (e.g., IFNy, ILip, TNFa) in the serum and in infiltration of human CDl lb + CD33 + CD16 + macrophages into the heart. Activated effector memory CD4 + and CD8 + T cells were observed in the periphery, which is detected by flow cytometric analysis.
  • cytokines e.g., IFNy, ILip, TNFa

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Abstract

Un procédé in vitro de préparation d'une population de cellules souches de mammifère hypo-immunitaires comprend la fourniture d'une population de cellules souches de mammifère isolées, les cellules souches de mammifère isolées exprimant une molécule d'adhésion cellulaire ; et la modification de l'expression de la molécule d'adhésion cellulaire dans la population de cellules souches de mammifère isolées pour diminuer ou inactiver l'expression de la molécule d'adhésion cellulaire et fournir la population de cellules de mammifère hypo-immunitaires. La population de cellules souches de mammifère isolées peut être des cellules souches pluripotentes, ou des cellules souches embryonnaires, et peut être des cellules souches humaines ou non humaines.
PCT/US2023/077747 2022-10-25 2023-10-25 Inhibition de molécules d'adhésion pour thérapies par cellules souches WO2024092015A1 (fr)

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Citations (4)

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Publication number Priority date Publication date Assignee Title
US10968426B2 (en) * 2015-05-08 2021-04-06 President And Fellows Of Harvard College Universal donor stem cells and related methods
US20210292715A1 (en) * 2018-07-17 2021-09-23 The Regents Of The University Of California Cells differentiated from immunoengineered pluripotent cells
WO2021222285A2 (fr) * 2020-04-27 2021-11-04 Sana Biotechnology, Inc. Dosage répété de cellules hypoimmunogènes
US20220213434A1 (en) * 2019-05-10 2022-07-07 The Regents Of The University Of California Modified pluripotent cells

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US10968426B2 (en) * 2015-05-08 2021-04-06 President And Fellows Of Harvard College Universal donor stem cells and related methods
US20210292715A1 (en) * 2018-07-17 2021-09-23 The Regents Of The University Of California Cells differentiated from immunoengineered pluripotent cells
US20220213434A1 (en) * 2019-05-10 2022-07-07 The Regents Of The University Of California Modified pluripotent cells
WO2021222285A2 (fr) * 2020-04-27 2021-11-04 Sana Biotechnology, Inc. Dosage répété de cellules hypoimmunogènes

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Title
YUKO KITANO: "Generation of hypoimmunogenic induced pluripotent stem cells by CRISPR-Cas9 system and detailed evaluation for clinical application", MOLECULAR THERAPY- METHODS & CLINICAL DEVELOPMENT, NATURE PUBLISHING GROUP, GB, vol. 26, 1 September 2022 (2022-09-01), GB , pages 15 - 25, XP093168046, ISSN: 2329-0501, DOI: 10.1016/j.omtm.2022.05.010 *

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